High Levels Of Toxins Research Articles

Phytotoxicity of Fumonisin B1, Moniliformin, and T-2 Toxin to Corn Callus Cultures

The phytotoxicity of the Fusarium moniliforme metabolite, fumonisin B 1 (FB 1 ), to corn callus cultures was determined and compared with two other Fusarium mycotoxins with known phytotoxic effects, moniliformin, and T-2 toxin. The callus was grown on modified MS medium containing either 0. 1, 1.0, 10, or 100 mg of toxin per liter. Callus growth was reduced as the concentration of FB 1 in the culture medium increased, resulting in a significant inhibition at 1.0 mg/ L (1.30 μM) and higher toxin levels (...)

Passive immunization of hamsters against disease caused by Clostridium difficile by use of bovine immunoglobulin G concentrate

Gestating Holstein cows were vaccinated with Clostridium difficile toxoid prepared from the culture filtrate of a strain that produces high levels of toxins A and B and other antigens. A bovine immunoglobulin G (IgG) concentrate was prepared from colostrum collected at parturition. The results of our studies showed that hamsters treated prophylactically with the hyperimmune bovine IgG concentrate were protected against C. difficile disease. These results suggest that orally administered hyperimmune bovine IgG specific for C. difficile culture filtrate may be useful in prophylaxis against C. difficile disease.

Protection against experimental pseudomembranous colitis in gnotobiotic mice by use of monoclonal antibodies against Clostridium difficile toxin A

The pathogenicity of Clostridium difficile is due to the production of two toxins (toxins A and B). We prepared monoclonal antibodies against toxin A and determined whether axenic mice passively immunized with the monoclonal antibodies were protected against C. difficile disease. The mice were kept in an isolator and were given ascites fluid intravenously prior to challenge with a toxinogenic strain of C. difficile. Control mice and mice receiving ascites fluid devoid of toxin antibody died within 2 days and had high levels of toxins A and B in their feces. Mice that received ascites fluid containing high amounts of toxin A monoclonal antibodies directed against the repeating units of the toxin survived. In protected mice, toxin B levels were similar to those in dying mice, but toxin A levels were greatly reduced. These data show that passive immunity induced by monoclonal antibodies against toxin A was effective against pseudomembranous cecitis.

Paralytic shellfish toxins seem absent in extracts of diarrhetic shellfish toxins

AbstractIt has been assumed that the water‐soluble paralytic shellfish toxins are extracted and concentrated by the organic‐solvent extraction method used for diarrhetic shellfish toxins. As diarrhetic toxin extracts of mussels with high levels of paralytic toxins are found nontoxic, it appears that paralytic toxins are not present in those extracts.

Microbiological Indicators of Subsurface Hydrocarbon Accumulations

The measurement of toxin tolerance in the microbial communities present in stream sediments has been demonstrated to be an effective exploration tool. The technique relies on the fact that metals of, or associated with, ore deposits often represent toxins to microbial communities. The microbes adapt to the presence of these toxins by genetic mutation of replicon structures called plasmids that then carry the coding that allows survival in their presence. The level of tolerance developed is proportional to the concentration of toxin present and shows measurable, statistically meaningful variance at levels normally below the detection thresholds of standard inorganic techniques. Hydrocarbons reaching the surface by macro- and microseepage, the metals adsorbed to them, and the geochemical changes they bring about in the near-surface environment also represent toxins or stresses to microbial communities that they must adapt to. Adaptation is again accomplished by genetic mutation and is measured by collecting a sediment sample, separating the microbial population from it, and exposing splits of the sample to several discrete concentrations of the hydrocarbon or metal being used as a seepage indicator. Tolerance to higher levels of toxin is interpreted to be indicative of the strength or volume of hydrocarbon seepage. The authorsmore » have undertaken this work using marine samples from the Gulf of Mexico because that setting has fewer surface geochemical variables than subaerial settings. Both gas and oil accumulations can be measured by this technique, and evidence for both macro- and microseepage has been found. Examples from the Mississippi Canyon area will be used to illustrate the efficacy of the technique.« less

Toxicity of puffers from Okinawa, Japan.

A total of 65 specimens of puffers comprising nine species of the suborder Tetraodontoidei were collected from Okinawa and tested for toxicity levels of the flesh, skin, liver, gonad, and intestine. Chelonodon patoca, Arothron manillensis, A. nigropunctatus, and A. hispidus showed high toxin levels in the flesh and thus should be banned from being eaten. Toxicities of the flesh of A. mappa, A. stellatus, Pleuranacanthus sceleratus, and Fugu niphobles were within an acceptable level. Diodon hystrix, which is widely consumed locally, showed no significant level of the toxin in all tissues tested. Extremely high toxicities of the flesh of C. patoca (520 MU/g) and A. nigropunctatus (184 MU/g) are worthy of special mention.

Plasma levels of cutaneous burn toxin and lipid peroxides in thermal injury

Lipid peroxides, formed as a consequence of oxygen free radical formation, are responsible for tissue damage in a great variety of pathological conditions including thermal injury. ‘Cutaneous burn toxin’, formed by application of heat to skin, is thought to be specific to the burn injury. It causes dose-dependent damage to mitochondrial and red cell membranes, and dose-dependent inhibition of interleukin-2-dependent growth of lymphocytes. The possibility that the toxicity of ‘cutaneous burn toxin’, a lipid-protein, is exerted through lipid peroxides, was examined by measuring the levels of both agents in plasmas of eight burn patients during the first week after their injury. It was observed that plasma lipid peroxides did not appear in parallel with absorption into the circulation of ‘cutaneous burn toxin’. Lipid peroxide levels equally common to very low and very high burn toxin levels, were recorded. The pair of agents correlated negatively ( r= − 0.26) at a significance of only 0.1. In addition, isolated purified ‘cutaneous burn toxin’ contained no measurable lipid peroxide. No relationship was therefore demonstrated between plasma levels of ‘cutaneous burn toxin’ and lipid peroxides.

Induction of aflatoxin biosynthesis inAspergillus parasiticus by ascorbic acid-mediated lipid peroxidation

The effect ofl-ascorbic acid on the biosynthesis of aflatoxin inAspergillus parasiticus was studied. Ascorbic acid at lower concentrations did not inhibit the growth of fungus but markedly induced aflatoxin biosynthesis. At a concentration of 1000 ppm of ascorbic acid, 4.8-fold higher levels of aflatoxin were detected. Copper did not enhance the induction of toxin synthesis by ascorbic acid when added to the growth medium. Ascorbic acid at 1000 ppm was also found to induce aflatoxin synthesis in resting mycelia. Chloroform (1% vol/vol) was found to induce aflatoxin synthesis under similar conditions. Ascorbic acid in the presence of ferrous ion can cause lipid peroxidation, which in turn is responsible for the induction of aflatoxin synthesis. During the induction of aflatoxin synthesis by ascorbic acid, the uptake of carbon source (acetate) was not affected. This observation suggests that on ascorbic acid treatment a precursor or an intermediate of aflatoxin biosynthesis is synthesized in vivo and is responsible for the higher levels of toxin without increasing the uptake of acetate.

Relationship between levels of Clostridium difficile toxin A and toxin B and cecal lesions in gnotobiotic mice

Various Clostridium difficile strains were studied with respect to their pathogenicity in monoassociated mice in relation to levels of toxin A and toxin B in vivo and in vitro. Two strains which were the most potent toxin producers in vitro induced mortality (100%); mice monoassociated with these strains were found to have high levels of both toxins in their ceca and an intense cecal epithelial ulceration together with a severe inflammatory process. No mortality was observed with the other strains. Strains which were moderately toxinogenic in vitro induced inflammation of the cecum but no ulceration, and no toxin A was found. Inflammation intensity was not related to toxin B levels. After 3 weeks, ceca returned to normal in spite of a chronic cytotoxin production. When compared with in vitro results, which showed a good correlation between the levels of the two toxins, toxin A amounts in vivo were found to be lowered relative to toxin B levels. The lack of detectable toxin A levels in animals infected with all but the two most highly toxinogenic strains prevented death. This work points out the importance of investigation of toxin A for the understanding of C. difficile pathogenicity.

Development and evaluation of enzyme-linked immunosorbent assays for detection of shiga-like toxin I and shiga-like toxin II.

Shiga-like toxin (SLT)-producing Escherichia coli has been associated with a spectrum of human illnesses, including hemorrhagic colitis and hemolytic uremic syndrome. It produces at least two antigenically distinct toxins designated SLT-I and SLT-II, which have been implicated in disease. Currently available toxin assays, however, are not suitable for most clinical or public health laboratories. In this study, we have developed two sandwich enzyme-linked immunosorbent assays (ELISAs) based on toxin-specific murine monoclonal capture antibodies and rabbit polyclonal second antibodies which are specific for SLT-I and SLT-II. The SLT-I ELISA detected 200 pg of purified SLT-I, and the SLT-II ELISA detected 75 pg of purified SLT-II. The types of SLT produced by 166 human and 54 animal isolates of E. coli that produced moderate to high levels of toxin were determined by the ELISA, and results were confirmed by cytotoxin neutralization assays. With the exception of results from three strains, the tests agreed on the types of toxin present. DNA probe assays of 86 of 87 isolates also agreed with the ELISA and neutralization results. Although the SLT-II ELISA was specific for the SLT-II variant produced by porcine edema strains, most of the isolates examined produced levels of toxin (less than 50 50% cytotoxic doses [CD50] per ml) below the detection limit of the test. The ELISAs were not sufficiently sensitive to consistently detect low levels of toxin (less than 50 CD50 per ml) found in fecal extracts. On the basis of these findings, both ELISAs appeared to detect significant levels of SLT-I ( > 100 CD50 per ml) and SLT-II ( > 50 CD50 per ml) in E. coli culture extracts and should be useful diagnostic tools in many microbiology laboratories.

Identification of toxS, a regulatory gene whose product enhances toxR-mediated activation of the cholera toxin promoter

We describe the cloning of the toxS gene from Vibrio cholerae E1 Tor strain E7946. This gene lies downstream from the toxR gene, which encodes the transcriptional activator for the cholera toxin (ctx) operon in V. cholerae. We show that ToxS acts in conjunction with ToxR to activate expression of the ctx operon in Escherichia coli. The classical strain 569B, which is attenuated for virulance but which synthesizes high levels of cholera toxin in vitro, carries a deletion of 1.2 kilobase pairs of DNA, downstream from the toxR gene, which removes toxS. We present evidence that toxS is the downstream gene in an operon with toxR.

Levels of toxic shock syndrome toxin-1 production among staphylococcus aureus strains and clinical implications

Among 250 S. aureus clinical isolates, the incidence of TSST-1 production was 18.0%. S. aureus var. hominis strains were predominant (95.6%), producing high levels of toxin in vitro, within the range of 0.6 to 4.3 micrograms/ml and exhibiting crystal violet binding with C/D pattern. No correlation was found between the level of TSST-1 production in vitro and the clinical course. Two (3.4%) of the var. bovis strains produced toxin in amounts less than 0.6 micrograms/ml and did not bind crystal violet. None of the 24 var. canis isolates produced TSST-1. Fifty five per cent of the isolates of phage group I produced TSST-1 and corresponded to 57.8% of the toxigenic strains. Two of the 250 patients developed toxic shock syndrome.

Staphylococcal alpha toxin promotes blood coagulation via attack on human platelets.

Staphylococcus aureus plays a major role as a bacterial pathogen in human medicine, causing diseases that range from superficial skin and wound to systemic nosocomial infections . The majority of S. aureus strains produces a toxin, a proteinaceous exotoxin whose hemolytic, dermonecrotic, and lethal properties have long been known (1-6). The toxin is secreted as a single- chained, nonglycosylated polypeptide with a M(r) of 3.4 x 10(4) (7, 8). The protein spontaneously binds to lipid monolayers and bilayers (9-14), producing functional transmembrane pores that have been sized to 1.5-2.0-nm diameters (15-18). The majority of pores formed at high toxin concentrations (20 mug/ml) is visible in the electron microscope as circularized rings with central pores of approximately 2 nm in diameter. The rings have been isolated, and molecular weight determinations indicate that they represent hexamers of the native toxin (7). We have proposed that transmembrane leakiness is due to embedment of these ring structures in the bilayer, with molecular flux occurring through the central channels (15, 19). Pore formation is dissectable into two steps (20, 21). Toxin monomers first bind to the bilayer without invoking bilayer leakiness . Membrane-bound monomers then laterally diffuse and associate to form non-covalently bonded oligomers that generate the pores. When toxin pores form in membranes of nucleated cells, they may elicit detrimental secondary effects by serving as nonphysiologic calcium channels, influx of this cation triggering diverse reactions, including release of potent lipid mediators originating from the arachidonate cascade (22-24). That alpha toxin represents an important factor of staphylococcal pathogenicity has been clearly established in several models of animal infections through the use of genetically engineered bacterial strains deleted of an active alpha toxin gene (25-27). Whether the toxin is pathogenetically relevant in human disease, however, is a matter of continuing debate. Doubts surrounding this issue originate from two main findings. First, whereas 60 percent hemolysis of washed rabbit erythrocytes is effected by approximately 75 ng/ml alpha toxin, approximately 100-fold concentrations are required to effect similar lysis of human cells (4-6, 13). The general consensus is that human cells display a natural resistance towards toxin attack. The reason for the wide inter-species variations in susceptibility towards alpha toxin is unknown but does not seem to be due to the presence or absence of high-affinity binding sites on the respective target cells (20, 21). Second, low-density lipoprotein (28) and neutralizing antibodies present in plasma of all healthy human individuals inactivate a substantial fraction of alpha toxin in vitro. These inactivating mechanisms presumably further raise the concentration threshold required for effective toxin attack, and it is most unlikely that such high toxin levels will ever be encountered during infections in the human organism. The aforegoing arguments rest on the validity of two general assumptions. First, the noted natural resistance of human erythrocytes to alpha toxin must be exhibited by other human cells. Second, toxin neutralization by plasma components, usually tested and quantified after their preincubation with toxin in vitro, must be similarly effective under natural conditions, and protection afforded by these components must not be restricted to specific cell species.

Three Trophic Level Interactions: Allelochemicals, Manduca sexta (L.), and Bacillus thuringiensis var. kurstaki Berliner

The role of plant allelochemicals in reducing the ability of Bacillus thuringiensis var. kurstaki Berliner to kill larvae of Manduca sexta (L.) was studied by comparing the effects of the alkaloid nicotine and the flavonoid rutin on the toxicity of B. thuringiensis. Concentrations of nicotine and rutin reflecting levels found in tobacco and tomato plants (0, 0.2, 0.4, 0.6, 0.8, 1.0% wet weight) were incorporated into synthetic diet containing 0.001% B. thuringiensis and B. thuringiensis -free diet. Survivorship of M. sexta larvae increased at higher levels of nicotine (0.6, 0.8, 1.0% wet weight), but higher concentrations of rutin did not increase larval survivorship. Experiments on colony growth of B. thuringiensis on nutrient agar containing nicotine or rutin (0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9% wet weight) demonstrated that nicotine reduced colony growth to a greater extent than rutin. Consequently, plant allelochemicals potentially are an important factor in reducing larval mortality caused by B. thuringiensis on certain crops. Specialist herbivores that can tolerate high concentrations of allelochemicals may gain protection from pathogens by feeding on plants or plant parts with higher levels of toxins.

Molecular evolution of pathogenic Escherichia coli.

Over the past four decades, Escherichia coli bearing certain of the 164 known somatic O antigens of the genus have been shown to be associated with intestinal infections. These strains may be termed enterovirulent E. coli (EVEC). Only a small percentage of the ^9000 0:H serotypes possible are known to be enteric pathogens. Detailed studies of the pathogenic mechanisms involved permit the separation of EVEC into four groups [1]: (7) enterotoxigenic E. coli (ETEC) that adhere to the mucosa of the small bowel and produce heat-labile (LT) toxin, heat-stable (ST) toxin, or both; (2) enteroinvasive E. coli (EIEC) that produce neither LT nor ST but, like Shigella, penetrate and multiply within the epithelial cells lining the colon; and (5) enteropathogenic E. coli (EPEC) that adhere to the mucosa of the small intestine and produce a characteristic effacement of microvilli. The latter are not invasive and do not produce LT or ST. The fourth group has been termed enterohemorrhagic E. coli (EHEC); this group causes hemorrhagic colitis and hemolytic uremic syndrome (HUS) and are of the serotype 0157:H7. In this issue of the Journal, Whittam et al. [2] present genetic evidence of a clonal descent of E. coli 0157:H7, the strain associated with hemorrhagic colitis and HUS. This report suggests that 0157:H7 is a newly evolved strain originating from a single ancestor. Until recently, 0157:H7 was a rare serotype that was seldom isolated from humans and animals. When isolated during outbreaks of hemorrhagic colitis, these strains have been shown to be neither invasive nor enterotoxigenic. This serotype also was not a recognized enteropathogenic serotype. However, 0157:H7 strains produce high levels of Shigalike toxin (SLT), which is related to Shiga toxin of Shigella dysenteriae type 1 [3]. These toxins also have

47] Immunochemical assays for toxic shock syndrome toxin-1

This chapter describes the immunochemical assays for toxic shock syndrome toxin-1 (TSST-1). The chapter discusses the assay to assess the capacity of tampons to alter TSST-1 production. (TSST-1) is a low-molecular-weight single peptide toxin secreted by some strains of Staphylococcus aureus. The toxin is highly associated with menstrually related toxic shock syndrome (TSS) and is considered as a causative toxin. An assay was developed based upon double immunodiffusion to quantify TSST-1 for the purpose of elucidating both conditions that yield high levels of toxin for ease of purification and the possible mechanism of tampon involvement in TSS. TSST-l-producing S. aureus are cultured in the desired medium, usually from 1 to 100 ml. Subsequently, 4 vol 4° absolute ethanol is added (it is not necessary to remove the cells), and the cultures placed at 4° for at least 1 hr to allow the TSST-1 to precipitate. The sensitivity of the assay may be increased 2- to 4-fold by drying and staining the slides.

Localization of stx, a determinant essential for high-level production of shiga toxin by Shigella dysenteriae serotype 1, near pyrF and generation of stx transposon mutants.

Hfr strains of Shigella dysenteriae serotype 1 were constructed by transient integration of an RP4 plasmid derivative carrying transposon Tn501 into the Shigella chromosome through Tn501-mediated cointegration. The Hfr strains were mated with Escherichia coli K-12 recipients carrying various auxotrophic markers, and E. coli recombinants which had received prototrophic Shigella genes were selected. Some of the E. coli transconjugants produced high levels of a cytotoxin which was neutralized by both polyclonal and monoclonal anti-Shiga toxin sera. The determinant for Shiga toxin production, designated stx, was first transferred to E. coli K-12 and then mapped by Hfr crosses to the trp-pyrF region located at 30 min on the E. coli chromosome. Bacteriophage P1-mediated transduction analysis of stx gave the following gene order: trp-pyrF-stx. The level of Shiga toxin production in E. coli Stx+ transconjugants and transductants was as high as that of the parental S. dysenteriae 1 strain. Stx- mutants of an Stx+ E. coli transductant were generated by random in vivo insertion mutagenesis with a Tn10 derivative transposon, Tn-mini-kan, followed by P1 cotransduction of the kanamycin resistance and PyrF+ markers into a pyrF Stx+ E. coli K-12 recipient. One stx::Tn-mini-kan transposon mutation was transferred by P1 transduction from this E. coli Stx- mutant to an E. coli K-12 Hfr strain and in turn transferred by conjugation to the original S. dysenteriae 1 strain plus two others. All kanamycin-resistant recombinants of S. dysenteriae 1 had lost their ability to produce high levels of Shiga toxin. A gene that specifies high-level Shiga toxin production is thus located near pyrF on the chromosome of S. dysenteriae 1. Stx- mutants of S. dysenteriae 1 exhibited full virulence in the Serény test.

Clostridium difficile--a spectrum of virulence and analysis of putative virulence determinants in the hamster model of antibiotic-associated colitis.

Each of nine different toxigenic strains of Clostridium difficile was administered orally to groups of hamsters pre-treated with clindamycin and housed individually in sterile isolator boxes. Faecal pellets and caecal contents from well, diarrhoeic, moribund and freshly dead animals were analysed for C. difficile and toxins A (enterotoxin) and B (cytotoxin), and tissue obtained when animals were killed was examined histologically. Not all strains were equally virulent in this model. Four strains of C. difficile killed all animals within 48 h and are designated as highly virulent for hamsters. These strains were clinical isolates from three cases of disease in man and one case in a hamster. Five strains caused death of some animals but only after 5 and upt to 13 days and are designated as less virulent for hamsters. These strains were isolated from asymptomatic infants (2) and household pets (2), and from the environment (1). The surviving test hamsters were killed after 14 days and, in most cases, were colonised by C. difficile, though levels of toxins A and B in caecal contents were low. None of the cultures used for challenge was capsulate or hydrophobic. There was no correlation between virulence and production of toxins A and B in vitro in tryptic-nitrate broth. With two strains examined, there was a correlation between virulence and toxin A (but not toxin B) production in caecal emulsions derived from clindamycin pre-treated hamsters. Caecal contents from the majority of moribund and freshly dead animals had quantities of toxin A sufficient to cause disease or death if given orogastrically. Toxin B was not produced in a fixed ratio with toxin A. The data support the view that high virulence of C. difficile is determined by efficient disease-inducing colonisation of the gut and the ability to generate, rapidly, high levels of toxin A in vivo.

Olfaction and hemodialysis: baseline and acute treatment decrements.

The effect of hemodialysis (HD) on olfactory recognition and memory function was investigated in people receiving chronic HD treatment. Fifteen subjects were given an olfactory recognition task 0.5 h before and 0.5 h after a dialysis session in counterbalanced order. Ten dialysis patients received a verbal recall task twice. Ten age-matched normal subjects received the olfactory task twice. Results were: (1) olfactory scores in the HD group were significantly lower than control subjects scores; (2) within the dialysis sample, olfactory identification scores were significantly lower after treatment than before, and (3) there were no parallel decreases in memory performance of the dialysis group after a HD treatment. We therefore conclude that those subjects receiving HD treatment demonstrate acute and chronic deficits in olfactory recognition which are unlikely to be due to fatigue, cognitive disequilibrium, anticoagulant treatment or high levels of uremic toxins.

Identification and Quantification of Ricin Toxin in Animal Tissues using ELISA

The purpose of this study was to provide a rapid, relatively inexpensive, reliable and straightforward method for the demonstration and quantification of ricin in animal tissues following an intramuscular injection. The use of radioiodinated ricin provided an indication as to where the highest levels of toxin might be found; the highly sensitive and specific use of antibodies in Enzyme Linked Immunosorbent Assays (ELISA) was employed as an alternative to the more expensive and more tedious procedure of radioimmunoassay. The injection site, lymphoid tissues and liver were found to contain detectable amounts of the toxin. In cases where administration of ricin were suspected, confirmation using biopsied tissue samples could enable appropriate remedial measures to be taken. Alternatively, in cases of suspicious deaths, post mortem tissues would be used to identify the presence of toxin in the body and could be of significant value to forensic diagnosis. The advantage of this method over that of immunocytochemistry is discussed.