First reported isolation from New Zealand pasture ofPithomyces chartarum unable to produce sporidesmin

A total of 676 isolates ofPithomyces chartarum, recovered from pasture at a single site, were examined for their ability to produce sporidesmin. Two isolates did not produce sporidesmin in levels detectable by HPLC despite their ability to spore profusely. This is the first report of sporulatingP. chartarum isolated from New Zealand pasture which does not produce sporidesmin.Abbreviations RCA rabbit chow agar     

Interaction of sporidesmin,a mycotoxin fromPithomyces chartarum,with lipid bilayers

Sporidesmin, a mycotoxin fromPithomyces chartarum is a hydrophobic molecule. It can therefore be easily incorporated in the cell membrane, where it is likely to cause changes in the bilayer organization and the properties of membrane proteins. In order to understand the redox behaviour of sporidesmin in a hydrophobic environment, we have investigated the effects of oxidized and reduced sporidesmin on the phase transition properties of bilayers and on the susceptibility of bilayers to pancreatic phospholipase A₂ (PLA₂). The changes induced by sporidesmin in the thermotropic phase transition profiles of dimyristoyl-sn-3-phosphatidyl choline (DMPC) bilayers were similar to those caused by solutes known to localize in the glycerol-backbone region of the lipid bilayer, suggesting a similar localization for oxidized and reduced sporidesmin. Neither form of toxin disrupt the bilayer or membrane organization even at relatively high mole fractions. At concentrations <10 mole% both forms partitioned equally well in the gel and liquid-crystalline phases, whereas at higher concentrations (30 mole%) reduced sporidesmin is preferentially localized in the liquid-crystalline phase. These effects of sporidesmin on the phase properties of DMPC vesicles were also reported by the fluorescence behavior of 10-pyrenedecanoic acid (PDA). The effects of oxidized and reduced sporidesmins on PLA₂ kinetics are consistent with their ability to perturb bilayer organisation.     

Photosensitization in cattle grazing on pastures of Brahciaria decumbens Stapf infested with Pithomyces chartarum (Berk. & Curt.) M.B. Ellis

Aspects of photosensitization in bovines grazing on pastures of Brachiaria decumbens Stapf infested with Pithomyces chartarum (Berk. & Curt.) M.B. Ellis infested all pastures 45(2):117-136, 1978. This paper reports experimental studies on photosensitization in bovines grazing on different pastures of Brachiaria decumbens Stapf in the "Cerrados" region (Planaltina, DF). Climatic conditions, zinc content and occurence of fungi on pastures were investigated. Pithomyces chartarum (Berk. & Curt.) M.B. Ellis infested all pastures examined. Photosensitization was observed in one animal maintained on a pasture of B. decumbens formed with seeds from Australia. Clinical and necropsy data were similar to those related in literature for sporidesmin-intoxicated animals. An isolate of P. chartarum and samples of bovine bile were assayed for sporidesmin presence.     

Liver enzyme changes in a guinea-pig model of facial eczema (sporidesmiotoxicosis)

Sporidesmin, a hepatotoxin from Pithomyces chartarum, is responsible for facial eczema in ruminants. In an attempt to clarify the biochemical processes supporting sporidesmin toxicity and response of the liver, haematology, plasma biochemistry and liver enzyme changes were monitored for 21 days in a model for facial eczema resulting from a single intraperitoneal injection of 2.8 mg/kg BW sporidesmin to guinea pigs. Most plasma disturbances were observed 8 days after administration and accounted for starvation, liver cytolysis, and cholestasis or liver enzyme induction. Alterations of hepatic enzyme activities were intense with a maximum increase on days 2 for alkaline phosphatases (ALP) and 8 for gamma-glutamyltransferase (GGT), and a maximum decrease on day 21 for aspartate aminotransferase (ASAT) and alanine aminotransferase (ALAT). Comparison of liver and plasma enzyme changes indicates that GGT was the most reliable and significant plasma indicator of sporidesmin-associated liver alterations. Moreover, this study points out the validity of the one-dose intoxicated guinea-pig model for research on sporidesmin biochemical toxicity and pathobiology of facial eczema.     

Biological control of sporidesmin-producing strains of Pithomyces chartarum by biocompetitive exclusion

The feasibility of using atoxigenic strains of Pithomyces chartarum for the biological control of toxigenic strains of P. chartarum was examined. Pasture, treated with atoxigenic strains of P. chartarum , contained up to 80% less sporidesmin than found in untreated pasture. Maximum sporidesmin levels of 26 ng g⁻¹ grass in treated pasture and 113 ng g⁻¹ grass in untreated pasture (means of 24 and four plots, respectively) were recorded 14 weeks after treatment, when spore numbers had reached a maximum of 80 000 spores g⁻¹ grass in the untreated plots and 50 000 spores g⁻¹ grass in the treated plots. This trial demonstrated that sporidesmin-producing spores of P. chartarum could be successfully reduced in pasture by the addition of atoxigenic strains, thereby reducing the risk of facial eczema in livestock.     

Selective inactivation of glutaredoxin by sporidesmin and other epidithiopiperazinediones

Glutaredoxin (thioltransferase) is a thiol-disulfide oxidoreductase that displays efficient and specific catalysis of protein-SSG deglutathionylation and is thereby implicated in homeostatic regulation of the thiol-disulfide status of cellular proteins. Sporidesmin is an epidithiopiperazine-2,5-dione (ETP) fungal toxin that disrupts cellular functions likely via oxidative alteration of cysteine residues on key proteins. In the current study sporidesmin inactivated human glutaredoxin in a time- and concentration-dependent manner. Under comparable conditions other thiol-disulfide oxidoreductase enzymes, glutathione reductase, thioredoxin, and thioredoxin reductase, were unaffected by sporidesmin. Inactivation of glutaredoxin required the reduced (dithiol) form of the enzyme, the oxidized (intramolecular disulfide) form of sporidesmin, and molecular oxygen. The inactivated glutaredoxin could be reactivated by dithiothreitol only in the presence of urea, followed by removal of the denaturant, indicating that inactivation of the enzyme involves a conformationally inaccessible disulfide bond(s). Various cysteine-to-serine mutants of glutaredoxin were resistant to inactivation by sporidesmin, suggesting that the inactivation reaction specifically involves at least two of the five cysteine residues in human glutaredoxin. The relative ability of various epidithiopiperazine-2,5-diones to inactivate glutaredoxin indicated that at least one phenyl substituent was required in addition to the epidithiodioxopiperazine moiety for inhibitory activity. Mass spectrometry of the modified protein is consistent with formation of intermolecular disulfides, containing one adducted toxin per glutaredoxin but with elimination of two sulfur atoms from the detected product. We suggest that the initial reaction is between the toxin sulfurs and cysteine 22 in the glutaredoxin active site. This study implicates selective modification of sulfhydryls of target proteins in some of the cytotoxic effects of the ETP fungal toxins and their synthetic analogues.     

In vitro and in vivo mutagenicity studies on sporidesmin, the toxin associated with facial eczema in ruminants.

Sporidesmin, a fungal toxin with widespread distribution within New Zealand, is thought to exert toxic effects through oxidative damage. The purified chemical was tested for its ability to cause point mutations in four strains of Salmonella typhimurium (TA98, TA100, TA102 and TA1537), in the presence and absence of exogenous metabolic activation. Although toxic effects were seen at concentrations exceeding 400 mu gl/plate, there were no significant increases in revertant colonies. In strain TA102, these results were not modified by the presence of glutathione. In AA8 Chinese hamster cells, sporidesmin acted as a potent clastogen, causing chromosomal breaks at concentrations as low as 3 ng/ml, where there was very little reduction in cell viability. Effects were primarily at the chromatid level, but some chromosomal events were also seen. Following low doses, the most common events were chromatid deletions and induction of double minute chromosomes. Interchange events occurred at concentrations of 10 ng/ml and above. The most common of these events was an incomplete chromatid interchange, although some examples of complete chromatid and chromosomal interchange were seen. These in vitro experiments were subsequently extended to an in vivo study of sporidesmin-induced lymphocytic micronuclei (MN) in sheep. In a double blind experiment, 5 sheep were treated with a single high dose of sporidesmin. Blood samples were taken from these, and from 5 untreated sheep, at various intervals before and after treatment. Peripheral blood lymphocytes cultures were harvested and scored for MN in cytokinesis-blocked cells, as a measure of clastogenic activity of sporidesmin in vivo. Following decoding, statistical analysis of the data revealed no significant differences between the MN levels in peripheral blood lymphocytes of sporidesmin-treated and untreated sheep. Although the possibility still exists that clastogenic effects could occur in other species, the data indicate that sporidesmin is not a clastogen in sheep, even though this species is highly susceptible to the toxic effects of sporidesmin.     

No difference found in ribosomal DNA sequences from physiologically diverse clones of Karenia brevis (Dinophyceae) from the Gulf of Mexico

Maximum growth rate and toxin content were significantly differentamong five strains of Karenia brevis isolated from Texas andFlorida when grown under identical culture conditions. Sequenceanalysis of the 18S rRNA gene and internal transcribed spacer(ITS) regions revealed, however, that all five strains wereidentical. Consequently, a clear genetic basis for physiologicalvariability among various geographical isolates of K. brevisfrom the Gulf of Mexico could not be assessed using these geneticmarkers. Both the ITS and 18S rRNA regions may be useful inspecies-specific probe selection. At the intra-specific level,however, an alternative marker will be needed to assess thediversity among K. brevis populations in the Gulf of Mexico.     

Regulation of in vitro Ptr-toxin Production by Pyrenophora tritici-repentis Isolates by Environmental Parameters and Accumulation of Ptr-toxin in Culture over Time

Previous work has demonstrated the production of a low molecular weight toxin (designated Ptr-toxin) by isolates of Pyrenophora tritici-repentis (Died.) Drechs., the cause of tan spot of wheat (Triticum aestivum). Before the mechanisms by which Ptr-toxin is produced are understood, conditions for optimal toxin production must be identified. This study defined glucose concentration, light and temperature for optimal fungal growth and accumulation of Ptr-toxin in a defined mineral salts-glucose liquid medium. The accumulation of Ptr-toxin in culture was monitored over a 21-day period. The growth of six P. tritici-repentis isolates increased significantly over the range of 0.05–5.0% glucose; however, the greatest accumulation of Ptr-toxin was observed at 1 and 2% glucose. Significant increases in fungal dry weights were observed at 23 and 30°C, although the effect of temperature on toxin production remains questionable. No significant influence of light on growth and Ptr-toxin production was observed. Ptr-toxin continues to accumulate in culture over the 21-day period tested in this study. The results of the present study reveal much about the specific isolates included but little information upon which generalizations may be based. As such, screening numerous P. tritici-repentis isolates for Ptr-toxin production under one set of conditions appears to be unreliable.     

Decarboxylase activity involved in methyl ketone production by Staphylococcus carnosus 833, a strain used in sausage fermentation

Outbreaks of Vibrio parahaemolyticus gastroenteritis in the United States (Texas, New York and Pacific Northwest) in 1997-98 emphasized the need to develop molecular methods for identification and differentiation of these organisms. When outbreak isolates were analyzed for the enterobacterial repetitive intergenic consensus sequences, the Texas and New York outbreak isolates had a specific 850-bp DNA fragment that was absent in Pacific Northwest isolates. The 850-bp polymerase chain reaction (PCR) product was found in isolates of serovar O3:K6, which have an unusual potential to spread and cause infections. To develop a specific molecular detection method for serovar O3:K6, the nucleotide sequence of the 850-bp product was determined. The GenBank blast analysis did not show homology with any known Vibrio spp. gene sequences. Two PCR primers were designed to specifically amplify the unique sequences from serovar O3:K6 isolates. Genomic DNA from 10 Texas, eight New York, and seven Pacific Northwest outbreak isolates of V. parahaemolyticus was assayed by PCR. Texas and New York isolates were positive in the PCR assay, giving a 327-bp PCR product as predicted; however, Pacific Northwest isolates were negative, indicating the absence of the target gene. Texas and New York isolates were all serovar O3:K6; the Pacific Northwest isolates were not. The primers were tested with other Vibrio spp. and other closely related species and no amplification of the 327-bp PCR product was found. The PCR method can be used to specifically identify O3:K6 V. parahaemolyticus isolates in less than 6 h.     

Metal complexes of sporidesmin D and dimethylgliotoxin, investigated by electrospray ionisation mass spectrometry

Electrospray ionisation mass spectrometry (ES-MS) has been used to probe the coordination chemistry of metabolites such as sporidesmin D (spdD), found in the saprophytic fungus Pithomyces chartarum, and the related bisdethiobis(methylthio)gliotoxin (dimethylgliotoxin, Megtx). SpdD forms complexes of the type [spdD+M(MeCN)] and [2spdD+M]+ (M=Cu, Ag) and, at higher cone voltages, [spdD+M]+. The bis(ligand) ion [2spdD+M]+ was observed at very high cone voltages, indicating it has appreciable stability; the proposed structure of this species has a four-coordinate metal ion with two bidentate spdD ligands, coordinated through their SMe groups. 1H NMR titrations of spdD with K+, Ag+ and Cu+ provided additional evidence for complex formation with the soft metals. SpdD forms only relatively weak complexes with Zn2+, Cd2+, Co2+ and Mn2+, in keeping with the known reduced tendency of these metals to form stable thioether complexes. ES-MS studies of Megtx showed similar results to spdD, with stable adducts formed with Cu+ and Ag+ ions. The X-ray crystal structure of spdD is also reported.     

Cyanotoxins from Black Band Disease of Corals and from Other Coral Reef Environments

Many cyanobacteria produce cyanotoxins, which has been well documented from freshwater environments but not investigated to the same extent in marine environments. Cyanobacteria are an obligate component of the polymicrobial disease of corals known as black band disease (BBD). Cyanotoxins were previously shown to be present in field samples of BBD and in a limited number of BBD cyanobacterial cultures. These toxins were suggested as one of the mechanisms contributing to BBD-associated coral tissue lysis and death. In this work, we tested nine cyanobacterial isolates from BBD and additionally nine isolated from non-BBD marine sources for their ability to produce toxins. The presence of toxins was determined using cell extracts of laboratory grown cyanobacterial cultures using ELISA and the PP2A assay. Based on these tests, it was shown that cyanobacterial toxins belonging to the microcystin/nodularin group were produced by cyanobacteria originating from both BBD and non-BBD sources. Several environmental factors that can be encountered in the highly dynamic microenvironment of BBD were tested for their effect on both cyanobacterial growth yield and rate of toxin production using two of the BBD isolates of the genera Leptolyngbya and Geitlerinema. While toxin production was the highest under mixotrophic conditions (light and glucose) for the Leptolyngbya isolate, it was highest under photoautotrophic conditions for the Geitlerinema isolate. Our results show that toxin production among marine cyanobacteria is more widespread than previously documented, and we present data showing three marine cyanobacterial genera (Phormidium, Pseudanabaena, and Spirulina) are newly identified as cyanotoxin producers. We also show that cyanotoxin production by BBD cyanobacteria can be affected by environmental factors that are present in the microenvironment associated with this coral disease.     

Production of cyclopiazonic acid by Aspergillus tamarii Kita.

Production of the mycotoxin cyclopiazonic acid by Aspergillus tamarii Kita is reported for the first time. Examination of 23 isolates of the fungus showed that 22 produced the toxin under the culture conditions utilized.     

Induction of Stomatal Closure by Helminthosporium maydis Pathotoxin

Helminthosporium maydis pathotoxin caused a rapid inhibition of photosynthesis in whole leaves of maize having Texas male-sterile cytoplasm but not in leaves having normal cytoplasm. Electron transport, phosphorylation, and proton uptake activities of isolated chloroplast lamellae, from either normal or Texas male-sterile cytoplasm leaves, were unaffected by addition of toxin to the reaction mixture. The toxin was found to have a direct effect on stomatal functioning. Rates of transpiration were inhibited in treated leaves at times slightly preceding the observed effects on photosynthesis. In studies with isolated epidermal peels from Texas male-sterile cytoplasm maize leaves, the toxin inhibited light-induced K⁺ uptake by guard cells. All effects of the toxin on Texas male-sterile cytoplasm maize tissues were found to be similar to the mode of action of abscisic acid.     

Murine Toxicity of Cochliobolus carbonum

Seventeen wild-type strains of the phytopathogenic fungus Cochliobolus carbonum, tested by intraperitoneal injection into mice, were lethal within 48 hr. The lethal effect appeared to be a toxic rather than an infectious process, because death occurred within 3 hr after injection of two of the isolates and heat-killed cultures were lethal. Assays of ascospore progeny from two crosses involving three isolates indicated that the toxic metabolites were under genetic control and quantitative regulation. Studies of the toxicological, cultural, and chemical characteristics of these three strains indicated that more than one murine toxin was present.     

Inhibition by sporidesmin of hepatocyte bile acid transport.

Exposure of isolated rat hepatocytes (approx. 2 x 10(7)--5 x 10(7) cells/10ml of incubation mixture) to 0.5 mg of the mycotoxin sporidesmin for 30--60 min at 37 degrees C produced loss of plasma-membrane microvilli with some disruption of organelle distribution in the sub-surface region. There was accompanying inhibition of [14C]cholate and [14C]taurocholate transport, but bile acid conjugation was not altered. Inhibition of cholate uptake was maximal after exposure of hepatocytes to sporidesmin for 1 min, and was not reversed by washing cells free of extracellular sporidesmin. N-Ethylmaleimide (0.1 mM) or dithiothreitol (1 mM) partially protected hepatocytes from sporidesmin inhibition of bile acid uptake. Significant protection was not given by other thiols or by zinc sulphate, cholesterol, ascorbate or alpha-tocopherol. The results are discussed in terms of sporidesmin action on cell membranes and the toxin's effect on bile secretion.     

Erythrogenic toxins A, B and C: Occurrence of the genes and exotoxin formation from clinical Streptococcus pyogenes strains associated with streptococcal toxic shock-like syndrome

We report the study of 53 clinical isolates of group A streptococci, all from patients with streptococcal toxic shock-like syndrome. The strains were analysed for the occurrence of the genes of erythrogenic toxins (pyrogenic exotoxins) types A, B and C and in vitro production of these toxins. In contrast to reports indicating that 85% of the toxic shock-like syndrome-associated isolates contained the erythrogenic toxin A gene, only 58.5% of our strains harboured this gene. The erythrogenic toxin C gene was detected in 22.6% of the isolates. Erythrogenic toxin A and erythrogenic toxin B were produced by 68.7% and 58.3% of the strains containing either gene. For all group A streptococci, irrespective of clinical association, the erythrogenic toxin B gene was detected in all the isolates tested. Thus, it is difficult to define a specific role for erythrogenic toxin B in toxic shock-like syndrome as there was no clear correlation between this disease and the presence of toxin genes. Our results suggest the existence of other pathogenic factor(s) produced by group A streptococci which may stimulate human peripheral T lymphocytes in a manner similar to that of erythrogenic toxins, thus explaining different observations in previous epidemiological genetic studies.     

Accumulation of Elsinochrome Phytotoxin does not Correlate with Fungal Virulence among Elsinoë fawcettii Isolates in Florida

Citrus scab caused by Elsinoë fawcettii is cosmopolitan in humid citrus-growing areas. We have previously demonstrated that production of non-host selective elsinochrome phytotoxin is a prerequisite for fungal full virulence and lesion formation. In this study we evaluated 71 field-collected isolates from Florida for pathogenicity and toxin production and found most of the isolates to be pathogenic to rough lemon, grapefruit and sour orange and able to produce elsinochromes in axenic culture. Elsinochromes were recovered, for the first time, from leaf lesions infected by 21 isolates, including four isolates that did not produce any measurable toxin in culture. There was no direct correspondence between the level of elsinochrome production in culture and virulence among the isolates. However, Elsinoë isolates failed to produce elsinochromes in culture and in planta were non-pathogenic to citrus hosts tested. Several isolates were non-pathogenic or only moderately virulent to the citrus hosts tested even though they could produce elsinochromes in culture, a phenotype not previously described in Florida.     

Effect of zinc on the toxicity of the mycotoxin sporidesmin to the rat

The toxicity of sporidesmin to rats fed diets containing either 4 ppm or 200 ppm Zn was determined. The high zinc diet doubled the LD₅₀ level for sporidesmin given as a single dose, but the LD₅₀ dose for rats on a zinc deficient diet was not significantly different from that for rats on a diet containing zinc at levels sufficient for normal growth. Subsequent trials demonstrated that large doses of ZnSO₄ given orally protected rats from sporidesmin poisoning. The protection was short lived as zinc needed to be given at or soon after sporidesmin challenge.     

Nivalenol production by<Emphasis Type="Italic">Fusarium poae</Emphasis>

Fusarium sp. were isolated from Swedish nivalenol containing grain and tested for toxin production. OnlyF. poae, 6 of 10 isolates, produced nivalenol. Highest production (44.7 μg/g) was obtained cultured on rice during 4 week at room temperature and under near UV-light. FiveF. poae isolates from other countries did not produce nivalenol but T-2/HT-2 toxin. One Swedish isolate produced both types of trichothecenes. Treatment with fungicides in aF. poae infected experimental field reduced the nivalenol concentration in the harvested grain.