Fungicidal activity of cecropin A.

Cecropin A (CA) fungicidal properties were explored. Nongerminated and germinated Aspergillus spp. and Fusarium spp. conidia were treated with CA. CA achieved complete lethality at < or = 25 microM (99 micrograms/ml) for germinating, but not nongerminating, conidia of Aspergillus spp. CA achieved total lethality for nongerminated and germinating conidia of Fusarium spp at 1.5 microM (6 micrograms/ml). MIC and minimal lethal concentration assays in buffered RPMI medium gave similar results.

252Cf plasma-desorption mass spectrometry of lipid A from Enterobacter agglomerans.

Endotoxins from gram-negative bacteria are believed to be causative agents of byssinosis, an occupational pulmonary disease associated with exposure to cotton dust in textile mills. Lipid A preparations from Enterobacter agglomerans, a gram-negative bacterium commonly found in cotton and cotton dust, have been analyzed using plasma-desorption mass spectrometry. Results indicate the existence of at least two lipid A types which differ only by the presence of an additional oxygen atom whose position has been localized to the acyloxyacyl ester-linked side-chain of the distal portion of the molecule. The lower molecular weight compound of the two structures has the same molecular weight and presumably the same empirical formula as a well-characterized lipid A from Salmonella minnesota. The mass spectra of lipid A compounds obtained from S. minnesota and E. agglomerans show strong similarities. Palmitoyl, hydroxymyristoyl, myristoyl, and lauroyl side-chains which are known to be present in the former are inferred from spectral evidence to be present in the latter.

Biophysical alteration of lung surfactant by extracts of cotton dust.

Byssinosis, a lung disease that can affect cotton mill workers, may be caused in part by lipopolysaccharides (LPS) from Gram negative bacteria. In vitro, LPS complexes with sheep lung surfactant (SLS). To determine whether LPS in extracts of cotton dust alters the biophysical characteristics of lung surfactant, aqueous extracts (1.0% w:v) of sterile surgical cotton (SSC) and a bulk raw cotton dust (1182DB) were prepared. Aliquots of the soluble extracts were incubated with SLS and studied by sucrose gradient centrifugation, surface tension analysis, and high pressure liquid chromatography (HPLC). The chromatography was employed to analyse for 3-hydroxymyristate (3-HM), a fatty acid indicating LPS. Also, purified Enterobacter agglomerans LPS and 3-HM as controls and as mixtures with SLS, were studied by HPLC. Sucrose gradient centrifugation showed that SLS-SSC, SLS-1182DB, and the SLS control had similar densities that differed from the remaining controls. The SLS-1182DB exhibited a floccule absent in the other samples. Surface tension values of SLS-SSC and SLS-1182DB differed significantly from all controls but only slightly from one another. 3-Hydroxymyristate was detected by HPLC in the 3-HM control, EA-LPS, SLS-EA-LPS, and SLS-1182DB, but not in SLS-SSC or the remaining controls. Apparently, 3-HM was below the HPLC detection range in SSC. The data indicate that LPS in the 1182DB, SSC and EA-LPS samples complexed with SLS. Floccule development in SLS-1182DB but not in SLS-EA-LPS suggests a further component(s) present in the bulk raw cotton dust, as well as LPS, which complexes with SLS. The data suggest that biophysical alterations to lung surfactant may play a part in the pathogenesis of byssinosis.

Factors influencing endotoxin concentrations on cotton grown in hot, humid environments: a two year study.

Cotton leaf, bract, fibre from opened bolls, and soil samples were collected weekly during two growing seasons (1984, 1985). Total and Gram negative bacterial populations were determined for each sample. Representative bacterial isolates were identified and endotoxin concentrations determined. For both years total and Gram negative bacterial populations on all sample types remained relatively stable until plant senescence. Afterwards, until plant death by frost, counts for all samples increased dramatically. Enterobacter agglomerans was the predominant species on leaf and bract, whereas the "all other" Gram negative bacterial species classification was the most common on fibre, with E agglomerans a close second. Senescence affected the occurrence of the species isolated. Statistical analysis partitioned by sample type showed strong correlations between endotoxin concentrations and certain bacteriological and environmental variables. The data suggest that in hot, humid environments the concentration of endotoxin on cotton leaf, bract, and fibre may be predicted by total and Gram negative bacterial counts, daily high temperature, and week after plant germination.

Enterobacter agglomerans lipopolysaccharide-induced changes in pulmonary surfactant as a factor in the pathogenesis of byssinosis.

Lipopolysaccharide (LPS) from Enterobacter agglomerans and pulmonary surfactant mixtures were centrifuged in discontinuous sucrose gradients to determine whether LPS bound to surfactant and examined in a Langmuir trough with a Wilhelmy balance to determine whether LPS altered the surface activity of surfactant. The LPS was found to bind to the surfactant and altered its surface tension properties. The binding of LPS to surfactant in the lung may change the physiological properties of surfactant and be a possible mechanism for the pathogenesis of byssinosis.

Seasonal variation in aerobic bacterial populations and endotoxin concentrations in grain dusts.

Settled and respirable grain dust samples were collected at intervals over a 16-month period at two grain terminals on the lower Mississippi River. Total and gram-negative aerobic bacterial (GNB) populations, as well as endotoxin concentrations, were studied. Plate counts indicated that no viable bacteria were found to be present in the respirable samples. Endotoxin concentrations ranged from 0.0 to 6.4 ng (0.0 to 7.9 ng/m3 air) per respirable sample, indicating that bacteria were once viable in dust of respirable size or contamination occurred from larger, non-respirable particles. Numbers of bacteria from settled dusts, both total and GNB, remained relatively stable; however, the genera of the GNB populations varied. Enterobacter agglomerans was the predominant species in warm months, but not in winter. Pseudomonas and Klebsiella species increased in winter and decreased in summer. Other genera found were Citrobacter and Serratia. Endotoxin concentrations in settled dust samples ranged from 1.7 to 5.6 ng/mg dust.

Mesophilic microorganisms and endotoxin levels on developing cotton plants.

Healthy cotton leaf, bract, fiber and soil were collected twice each week during growing season. These samples were studied to determine the epiphytic total and gram-negative bacterial (GNB) populations and endotoxin levels from seedling development to harvest. Since bract is a significant trash component of raw cotton, a study of the epiphytic bract fungi was performed also. Bract and soil had the highest total bacterial count (TBC) until senescence, when the bract count increased significantly over that of the soil. Leaf TBC was usually third while fiber had the least TBC. Leaf senescence did not result in an increase in TBC as with bract. GNB counts paralleled, but were approximately 10-fold lower than that of the TBC. Enterobacter agglomerans was the most predominant bacterium on leaf and bract. Gram-positive rods were the most common bacteria on fiber with E. agglomerans second. Gram-positive rods were by far the predominant species in soil. Senescence affected the population levels of the various genera on leaf and bract. Endotoxin varied greatly for all sample types. Soil usually gave the highest amounts, followed by bract, fiber leaf surface. Statistical analysis showed some minor correlations between the presence of certain bacteria with other bacteria. No correlations were found between weather data and bacterial types present or endotoxin levels. The epiphytic fungal population on bract was studied through the growing season. Though different genera appeared, the predominant genus throughout the study was Cladosporium.

Lectin grouping of Bacillus thuringiensis serovars.

Twelve lectins, 11 of plant and 1 of animal origin, were tested against the 28 serovars of Bacillus thuringiensis to study their agglutinating specificities. Except for the sialic specific lectin from Limulus polyphemus, tube agglutination assays were performed using lectin concentrations of 10, 50, and 100 micrograms against 0.2 mL of 10(9) cells/mL in a final volume of 1.0 mL. The agglutination studies with the Limulus lectin were performed using 10 and 50 micrograms of lectin. Tubes were incubated overnight at room temperature (25 degrees C) and observed for agglutination patterns. Ten of the 28 serovars were individualized according to their ability to bind with various lectins. The study shows that the various serovars have different carbohydrate residues which indicates that the O-somatic antigens differ.

Naphtho-gamma-pyrone production by Aspergillus niger isolated from stored cottonseed.

Aspergillus niger was found to be the predominant fungal contaminant of stored cottonseed. Seven strains were isolated and grown on rice. The hexane-insoluble material from methylene chloride extracts of 2-week-old cultures contained components toxic to mice. Based on high-pressure thin-layer and liquid chromatographic analyses, the major components in the mixture were eight different naphtho-gamma-pyrones. Of these, the hydrated dimeric naphthopyrones aurasperones B and C occurred in higher yield than aurasperones A, iso-A, and D and the monomeric naphthopyrones flavasperone and rubrofusarin, all of which were present in the mixture. In addition, fonsecin monomethyl ether was isolated. This metabolite may be a precursor in the biosynthesis of the hydrated aurasperones; it has not been identified previously as a metabolite of A. niger. The relative amounts of the different naphthopyrones were dependent on both the growth substrate and the fungal isolate.

Gram-negative bacterial endotoxins in grain elevator dusts.

Twenty settled and 69 respirable grain dust samples were collected from 5 grain elevators along the lower Mississippi River. Studies were performed on 2-keto-3-deoxyoctonate (KDO) concentrations, total bacterial plate counts, gram-negative bacteria (GNB) count, identification of GNB, and endotoxin levels. KDO values were 52.1-510.0 ng/mg. A new method involving gas liquid chromatography was developed to analyze for KDO. No correlation could be made between KDO and endotoxin levels. Total bacterial plate counts for settled dusts ranged from 1.9 to 53.4 million/g, while the GNB count was 0.1-50.0 million/g. Two hundred and thirty GNB were isolated from all samples and identified. Sixty-one percent were Enterobacter agglomerans, followed by species of Pseudomonas (9.1%), Serratia (6.9%), Actinetobacter (6.9%) and other genera (16.2%). All but four of the airborne samples had no significant bacterial population. Twenty-three of sixty-nine airborne samples contained 0.6 ng of endotoxin while 3 filters had 6.0 ng. All settled dust samples were contaminated with endotoxin at levels ranging from 22.5 - 187.5 ng/mg. Based upon airborne endotoxin levels worker exposure range was extrapolated to be 0.0-0.2 ng/kg body weight per 8 hr period. This is much less than that necessary to elicit febrile and pulmonary responses. However, the heavily laden filters corresponded to an endotoxin dose of 1.6 ng/kg body weight.

Preliminary study of mycoflora and mycotoxins in grain dust from New Orleans area grain elevators.

Dust particles arising from disintegration of grain during handling and shipping may be inhaled by exposed barge and elevator workers. Since the normal grain mycoflora usually contain mycotoxin-producing fungi, these toxins could be natural contaminants of grain dust. Known mycoflora of commercial grain include species of Penicillium, Aspergillus, and Fusarium-fungi that may produce ochratoxins, aflatoxins and zearalenone. A procedure was developed to extract simultaneously these toxins from grain dust. Initial extraction with methylene chloride and water was followed by specific cleanup procedures for each toxin and then by thin layer chromatographic quantitation. In 50-g samples the lowest level of detection for zearalenone was 50 ng of zearalenone/g of dust, for ochratoxin, 10 ng/g and for aflatoxin, 5 ng/g. Dust samples were collected at various locations in two grain elevators and their associated transfer facilities in the New Orleans area. Large amounts of dust that had settled upon floors, machinery and ledges were tested, as well as that which had been collected by dust control systems. Samples were analyzed for aflatoxins, ochratoxin A, and zearalenone. None of the 15 samples contained any detectable amount of aflatoxins or ochratoxin A, but 10 of the 15 samples contained zearalenone at levels from 25 to 100 ng/g.

The inability of Fusarium species extracts to induce dieback in pine.

Cultures of F. moniliforme var. subglutinans, F. moniliforme, F. lateritium, F. equiseti, F. semitectum and F. solani from pine and F. moniliforme and F. graminearum from southern U.S. corn were grown on rice and corn, extracted, and checked for toxicity in mice, chicken embryos, and pine seedlings, and for mutagenicity by the Ames test. While extracts from both fungal groups contained toxins, none of the extracts induced dieback in pine seedlings. Almost all of the cultures isolated from corn in contrast to those from pine, were mutagenic. Thin-layer chromatography did not detect T-2 toxin, moniliformin, or vomitoxin, indicating that these toxins do not elicit dieback symptoms in pine.

Bacillus thuringiensis distribution in soils of the United States.

During a 2-year study, samples of various types of soils were collected from 115 fields that had not previously been tested with Bacillus thuringiensis and which were remote from any large-scale aggregations of lepidopterous insects in rearing or grain-storage areas. An average of about 400 isolates were examined from each soil, and, of 46 373 isolates examined, only 250 (0.5%) were identified as B. thuringiensis. While it was almost impossible to insure that a field had never been treated with B. thuringiensis or that drift from some nearby application had not reached the field, it is noteworthy that of the 250 isolates, 156 (62.4%) were not var. kurstaki, the only variety that has been used commercially in the United States in about 10 years. This is a strong indication that the B. thuringiensis isolates observed were present naturally. To verify the procedures used, samples were taken from two adjacent experimental plots which had been treated about 12 months previously with formulations of var. kurstaki and var. galleriae, respectively. With practically no exception, the variety recovered from each plot was the variety applied, indicating that the varietal status of B. thuringiensis is stable in the soil.

Role of lactone ring of aflatoxin B1 in toxicity and mutagenicity.

Cleavage of the lactone ring of aflatoxin B1 results in a nonfluorescent compound that has greatly reduced biological activity. Mutagenicity, as measured by the Ames test, is reduced 450-fold compared to that of B1, and toxicity, as measured by the chick embryo test, is reduced 18-fold.

Storage Quality of Brown Rice as Affected by Packaging with and without Carbon Dioxide.

Shelled brown rice (100 g/bag) was packed in regular plastic bags in air, in laminated film (nylon-EVA) bags in air, and in laminated bags plus CO2. Samples of each were stored in the dark at 4 C and at 24 C, and samples were removed after 1, 3, 5 and 7 months for analysis of odor changes, free fatty acids, total microbial counts, total lipolytic fungi and bacteria, lipid peroxides and gas chromatographic volatiles profiles. Brown rice in laminated bags plus CO2 was more stable under refrigerated conditions than at ambient temperatures. However, at 24-C storage there was no consistent significant decrease in free fatty acids, lipid peroxides and volatile compounds in these bags compared to the other types of packing. Laminated bags seem to have had an adverse effect on total microbial populations at both 4 and 24 C. Selection of either type of package for brown rice would be governed by end use, storage time and conditions.

Production of Aflatoxins B and G on Solid and Broth Culture Media.

Mycelial growth and production of aflatoxins by Aspergillus parasiticus NRRL A-16,462, on whole peanuts, undelintered cottonseed, and shredded wheat was compared to growth and aflatoxin production on 10% broths of the same substrates. The greatest amount of toxins was produced on shredded wheat as a whole substrate. The least amount was produced in shredded wheat broth medium. Intermediate levels of toxin were produced on cottonseed in both types of media. More aflatoxin was produced on peanut broth than on whole peanuts. Mycelial growth on whole shredded wheat was greater than growth on the other solid media though it had the lowest amount of growth of the three broth media. Among whole substrates, growth on fuzzy cottonseed was least but mycelial growth on cottonseed broth was second of the three broth media. Growth on whole peanuts was as great as on shredded wheat; on peanut broth, it was the greatest of the three broth media. On whole substrates, the ratio of aflatoxins B:G produced was highest on shredded wheat and lowest on peanuts. In broth culture the B:G ratio was largest on the cottonseed and lowest on shredded wheat.