Mycotoxins in fungal contaminated samples of animal feed from western Canada, 1982-1994.

Feed samples from 94 cases involving fungal contamination and suspected mycotoxicosis of farm animals in western Canada were examined during 1982-1994 to assess the incidence of mycotoxins. Samples were analyzed for aflatoxins, ochratoxin A, citrinin, sterigmatocystin, and the fungal estrogen zearalenone. Samples infected with Fusarium fungi were additionally assayed for nivalenol, deoxynivalenol, fusarenone-x, 15-acetyl-deoxynivalenol, diacetoxyscirpenol, HT-2 toxin, and T-2 toxin. Mycotoxins were found in 21 feed samples from 17 cases (18% of the reported cases), generally at levels far below those needed to induce symptoms under laboratory conditions. HT-2 toxin and other type-A trichothecenes were detected in 5 samples, deoxynivalenol and other type-B trichothecenes in 13, ochratoxin A in 5, and citrinin in 2. In 9 cases, symptoms observed in the animals were consistent with the known effects of the mycotoxin(s) found in the particular feed samples.

Effect of medicating ingredients and other additives on microflora present on swine and chicken feeds from a Manitoba mill.

The occurrence of microfloral components on medicated and non-medicated swine and chicken feed pellets and crumbles, produced in a Manitoba feed mill between June 1991 and October 1992, was determined. Addition of medicates to feeds generally decreased bacterial incidence and increased that of Eurotium spp. fungi. The effect was less pronounced when copper sulphate was added to medicated swine feeds.

Nephrotoxigenic Penicillium species occurring on farm-stored cereal grains in western Canada.

The incidence of nephrotoxigenic Penicillium species on farm-stored cereals in western Canada was determined by morphological and metabolite profile examination. Of the 142 isolates examined 102 were toxin producers with 61 P. aurantiogriseum and 27 P. freii. Other nephrotoxigenic species included P. tricolor (6 isolates), P. verrucosum Chemotype II (4 isolates) and P. viridicatum Westling (4 isolates). The nephrotoxigenic Penicillium species profile for western Canada appears to differ from that of Denmark where P. verrucosum, P. cyclopium, P. freii and, to a lesser extent, P. aurantiogriseum, P. polonicum, and P. viridicatum predominate.

Identification of NephrotoxicPenicillium Species from Cereal Grains.

A system is described for identifying grain-inhabiting nephrotoxicPenicillium spp. based on their colony characters on Czapek yeast extract agar, yeast extract sucrose agar, and malt extract agar media, and their secondary metabolite profiles on thin layer chromatography plates. Using this system, the identity of 11Penicillium species, or their chemotypes, producing nephrotoxic metabolites could be confirmed. The species areP. verrucosum chemotype I, P.verrucosum chemotype II,P. expansum, P. citrinum, P. aurantiogriseum, P. freii, P. tricolor, P. polonicum, P. viridicatum, P. cyclopium, and P. melanoconldlum. Other non-nephrotoxicPenicillium species present on stored grains were separated from nephrotoxic species by their colony characters and metabolite profiles.

Mycotoxin production in amber durum wheat stored at 15 and 19% moisture content.

Ochratoxin A and citrinin developed in 11 kg parcels of amber durum wheat at 15% and 19% initial moisture content (MC) exposed to simulated bulk storage in a Manitoba granary for 60 weeks between July 1984 and September 1985. Other biotic and abiotic variables were monitored throughout the storage period. Ochratoxin A reached maximum levels of 11.8 and 0.11 ppm at 19 and 15% initial MC, respectively, during weeks 44-48; citrinin reached levels of 80.0 and 0.65 ppm at these respective moistures during the same period. The effect of 19% initial MC was significantly greater for the following variables: ochratoxin A, citrinin, Aspergillus flavus, Aspergillus glaucus group species, Alternaria species, Aspergillus versicolor, bacteria, fungal propagule count, seed germination, O2, CO2, moisture content, and fat acidity. Principal component analysis indicated that the first two components, describing greater than 60% of the variability in the data, partially defined the ecological relationships leading to mycotoxin production in the stored durum wheat system.

Mycotoxin formation in HY-320 wheat during granary storage at 15 and 19% moisture content.

Eleven-kilogram parcels of HY-320 wheat, a cultivar of the new Canada Prairie Spring class, were kept at 15 and 19% initial moisture contents (IMC) in simulated storage in a Manitoba farm granary for 60 weeks to determine biotic and abiotic changes and mycotoxin production. Ochratoxin A reached a maximum of 0.24 ppm by week 20 in the 19% IMC wheat, but was absent in the 15% IMC wheat; no other mycotoxins were detected. Temperature, moisture content, O2 and CO2 levels, fat acidity values, seed germination, microfloral incidence and abundance, and the presence of other mycotoxins were monitored. Principal component analysis of all variables showed that the first principal components accounted for 32-41% of the system variability, and contained the ochratoxin A variable. Ochratoxin A was produced in moist grain that had decreased seed germination and Alternaria activity, and high fungal activity by Penicillium and Aspergillus versicolor. Compared to other stored cereals previously studied, HY-320 wheat would be ranked in a low-risk category for mycotoxin formation, based on the ochratoxin A levels observed.

Mycotoxins and toxigenic fungi on cereal grains in western Canada.

Toxins occasionally present on cereal grains in the field in western Canada include ergot alkaloids produced by Claviceps purpurea and trichothecenes produced by Fusarium species, particularly Fusarium sporotrichiodes and Fusarium graminearum. HT-2 toxin, T-2 toxin, diacetoxyscirpenol, and deoxynivalenol are the main trichothecenes encountered. During storage of cereals, the predominant toxins and toxigenic fungi are ochratoxin A and citrinin produced by Penicillium aurantiogriseum, P. chrysogenum, and P. verrucosum and sterigmatocystin produced by Aspergillus versicolor. The incidence of toxin-contaminated grains is extremely low relative to the volume of grains produced. Occurrence of toxins is influenced by field moisture, temperature, and bin storage conditions of a particular year. The risk of toxin production is highest in durum wheat and lowest in oats.

Effect of magnesium on production of toxic-shock-syndrome toxin-1: a collaborative study.

We performed a series of collaborative experiments to clarify the effect of Mg++ on production of toxic-shock-syndrome toxin-1 (TSST-1) in various culture media. TSST-1 production was enhanced by adding ethylenediamine-tetraacetic acid (EDTA) at appropriate concentrations to brain-heart infusion and beef-heart medium. The magnitude of this effect depended both on the number of bacteria used to inoculate the media and on the sampling time. Large inocula prepared in media containing high levels of Mg++ introduced sufficient Mg++ to the growth medium to influence subsequent bacterial multiplication and toxin production. Small inocula of bacteria washed in Mg++-deficient medium before inoculation did not, however, multiply or produce toxin in Mg++-deficient medium. Maximal toxin expression occurred during late logarithmic phase, regardless of Mg++ concentration, and Mg++ appeared to control when late logarithmic stage of growth would be achieved. The toxin-enhancing effect of EDTA was reversed by adding excess Mg++ to treated medium.

Mycotoxin formation in moist 2-row and 6-row barley during granary storage.

Eleven-kilogram parcels of 2-row and 6-row barley initially at 18% moisture content were implanted in dry bulk oats in a farm granary in Manitoba for 60 weeks between August 1983 and October 1984. Temperature, moisture content, O2 and CO2 levels, fat acidity values, seed germination, microfloral incidence and abundance and the presence of major mycotoxins (aflatoxins, sterigmatocystin, ochratoxin A, citrinin, penicillinic acid, patulin) were monitored. Ochratoxin A reached maximum levels of 0.97 ppm by week 24 in the 6-row barley, and 0.05 ppm by week 28 in the 2-row; no other mycotoxins were detected. The effect of cultivar type was significant (P less than 0.01) with greater effects in the 6-row barley for the following parameters: fat acidity value, germination, incidence of Alternaria alternata, Penicillium spp. and Helminthosporium sativum, total fungal propagule count and ochratoxin A levels. The effect of time was significant (P less than 0.05) for all variables except oxygen, carbon dioxide, Aspergillus versicolor, and total fungal propagule count. The interaction between cultivar and time was significant (P less than 0.01) for Alternaria and Helminthosporium only.

Regulation of staphylococcal toxic shock syndrome toxin-1 and total exoprotein production by magnesium ion.

The effect of Mg2+ on in vitro production of extracellular proteins and, specifically, of toxic shock syndrome toxin-1 (TSST-1), by Staphylococcus aureus in a chemically defined medium was examined. As previously observed, the organisms did not proliferate in the absence of divalent cations. Low levels of Mg2+ (0.02 to 0.04 mM) permitted submaximal proliferation and elevated production of exoproteins. When the Mg2+ concentration was raised to 0.4 mM, multiplication was optimal and exoprotein levels were depressed. Ca2+ and Mn2+ diminished the effect of limiting Mg2+. The increased levels of exoproteins were not due to cell lysis or leakage since intracellular TSST-1 levels were not high enough to account for the increase in extracellular TSST-1 and since the intracellular enzyme, lactate dehydrogenase, was not found in culture supernatants. Cells cultured in low levels of Mg2+ remained in logarithmic growth longer than did those cultured in high concentrations of Mg2+ and, unlike the latter, produced exoproteins throughout the logarithmic growth phase. Low Mg2+ had no effect on cultures in the stationary phase, and organisms cultured in low Mg2+ recovered fully when transferred to high Mg2+. We conclude that, when cultured in medium deficient in Mg2+, S. aureus responds early in the growth cycle by increasing production of many extracellular proteins, including TSST-1.

Interrelations Among Ecological Variables in Stored Cereals and Associations with Mycotoxin Production in the Climatic Zones of Western Canada.

The incidence of mycotoxins and the interrelations among ecological variables in western Canadian common and durum wheat and barley were determined using 440 railway car samples collected during 1981-83. The 41 Prairie Crop Districts represented by the samples were ranked according to the incidence of fungal infection, mite infestation and other grain quality loss critera and grouped according to climatic subdivisions. Principal component analyses determined linear relationship patterns of ecological variables; ranking of the crop districts was done by Kendall's ranking approximation technique using the first (C1) and second (C2) principal components. Only five samples originating from Saskatchewan, Alberta and Manitoba contained ochratoxin A, with levels of 10-51 ppb. None of the samples contained aflatoxins, sterigmatocystin, citrinin, or penicillic acid. All samples containing ochratoxin A had established contamination of species in the Aspergillus glaucus group (10-86% infection level), and Penicillium spp. (20-80% infection level). Most of these samples had low germinability, high fat acidity levels and were infested by stored-product mites including, Acarus siro complex, Lepidoglyphus destructor (Shrank) and Tarsonemus granarius Lindquist. In wheat, the C1 accounted for 24% of the variability indicating that poorly germinated wheat was associated with the presence of Penicillium , Aspergillus glaucus group, Wallemia sp., and the fungivorous mites, T. granarius and A. siro . The C2 accounted for 10% of the variability indicating that an increase in free fatty acids was correlated with a high incidence of Aspergillus flavus and A. versicolor . Pronounced C1 interrelations for wheat were most common in crop districts lying in the Sub-humid Prairie, the northern part of the Dry Belt and the southern part of the Humid regions. Similar relationships for durum wheat and barley were also defined and ranked on maps.

Competitive, enzyme-linked immunosorbent assay for toxic shock syndrome toxin 1.

We developed a competitive, enzyme-linked immunosorbent assay for the quantitation of toxic shock syndrome toxin 1 (TSST-1). Polyvalent immunoglobulin G from immunized rabbits was used as the capture antibody, and alkaline phosphatase conjugated to purified toxin served as the indicator enzyme. A standard curve was generated with each experiment, from which the concentration of toxin in culture supernatants was extrapolated. The assay was useful for determining toxin concentrations of 0.03 to 0.5 micrograms/ml, which is a substantial, practical improvement over immunodiffusion methods. Staphylococcal enterotoxins A through E were not significantly cross-reactive in the assay, and staphylococcal protein A did not interfere with quantitation of TSST-1. By testing a variety of staphylococcal strains, we found 100% concordance between toxin determinations made with our assay and those made by the investigators from whom the strains were obtained. The competitive, enzyme-linked immunosorbent assay is a highly reproducible, inexpensive means of determining TSST-1 concentrations and may have broad applicability in the field of toxic shock research.

Plasma membrane biogenesis in eukaryotic cells: translocation of newly synthesized lipid.

We examined the transfer of sterols and phospholipids from their site of synthesis to the plasma membrane of Acanthamoeba castellanii. Cells were labeled with [3H]acetate, and plasma membrane fractions were isolated under conditions that minimize the nonspecific exchange of lipids between subcellular membrane fractions. Sterols and phospholipids were purified from both whole-cell homogenates and isolated plasma membrane. In whole cells, 3H-labeled lipids were formed, with no apparent time lag, in a linear manner up to 1 hr. Labeled sterol and phospholipids appeared in the plasma membrane, after a 30-min lag, at approximately the same rate. However, the ratio of newly synthesized sterol to phospholipid was significantly enriched in the plasma membrane relative to the whole cell, even at the earlier time points. Pulse-chase experiments indicated that sterols and phospholipids are turned over in the plasma membrane with similar, rather short half-lives. The results of these studies suggest that, although sterols and phospholipids are transported to the cell surface with similar kinetics, some sorting of the lipids must occur at an early stage in membrane biogenesis. The data are consistent with a model of lipid translocation by vesicular transport.

Mycotoxins and mycoflora in animal feedstuffs in western Canada.

Feed samples associated with 51 cases of suspected or potential mycotoxicoses of farm animals in western Canada were examined during a three year study. Ochratoxin A was detected in four cases, T-2 toxin and diacetoxyscirpenol in one, and sterigmatocystin in one. Samples examined for microflora associated with production of these mycotoxins contained Penicillium spp., Aspergillus ochraceus, Fusarium spp. and fungi of the Aspergillus glaucus group. Samples were analyzed for T-2 toxin and diacetoxyscirpenol only if Fusarium spp. were present. The first known incidence of suspected sterigmatocystin poisoning of poultry through feed ingestion has been encountered.

Mycotoxin formation in moist wheat under controlled temperatures.

One-kilogram parcels of wheat with 20.5% moisture content were maintained at 15 degrees and 22 degrees C for 10 weeks to study quality changes. Temperature, moisture, oxygen and carbon dioxide levels, microflora incidence and abundance, seed germination, fat acidity values, aflatoxins, sterigmatocystin, ochratoxin A, penicillic acid, citrinin and zearalenone were monitored. By two weeks, trace levels of ochratoxin had formed at both temperatures. By 10 weeks, the wheat contained at least three times more ochratoxin A at 22 degrees C than at 15 degrees C. Strains of Penicillium verrucosum var. cyclopium were associated with ochratoxin A production. No other mycotoxins were detected. The effect of temperature was significant for all variables (greater effect at 22 degree C) except A. glaucus gr. and Penicillium (P less than .01). The effect of time was significant for all variables except bacteria (P less than .01). The shape of the response was fully characterized by the linear and quadratic terms, except for % moisture which was linear only, and for bacteria for which time was not significant. The interaction between time and temperature was significant (P less than .01) for total fungal propagule count, % moisture, and Aspergillus versicolor, indicative of the steeper rise in slope for 22 degrees C.

Perceived loudness and visually-determined auditory distance.

Three experiments were conducted to determine whether variations in the perceived distance to a test sound could influence its loudness in the absence of physical changes in sound-level. The phenomenon of visual capture provided the means for manipulating apparent distance. A 'dummy' loudspeaker was used to vary the apparent source location of a short noise stimulus while the true source of this sound remained fixed (and hidden) with respect to the observer. Sound-levels from 40 to 75 dB(A) were presented to independent groups of observers in either anechoic or semi-reverberant acoustical environments. In general, reported loudness increased with perceived distance. This finding has implications for conceptualizing the phenomenon of loudness constancy.