Aflatoxin B1 Induction of Lysogenic Bacteria

A technique for biological verification of aflatoxin B(1) was developed based on toxin-mediated induction of lysis in a lysogenic strain of Bacillus megaterium NNRL B-3695. Reduction of culture turbidity was determined at various concentrations of toxin. Incubation of 1.1 x 10(-4) g (dry weight) of cells/ml of growth medium containing 25 mug of B(1) per ml at 37 C reduced initial turbidity 0.20 absorbance units in 4 hr. If the bacterial lysate of the lysogenic strain, after a 2-hr incubation with 25 mug of B(1) per ml, was plated with a sensitive B. megaterium strain (NRRL B-3694), plaque-forming units increased approximately 150 times relative to the control. Comparable testing of the effects of aflatoxin on the nonlysogenic, sensitive strain demonstrated that 75 mug of B(1) per ml neither induced lysis nor plaque-forming units. Although induction is not an exclusive property of aflatoxin B(1), the differential response of the lysogenic and sensitive Bacillus strains to B(1) offers a unique and rapid technique for biological verification of the toxin.     

Mouse bioassay for Fusarium metabolites: rejection or acceptance when dissolved in drinking water.

Ten metabolites of Fusarium species (butenolide, diacetoxyscirpenol, equisetin, fusaric acid, gibberellic acid, moniliformin, NRRL 6227 peptide, T-2 toxin, vomitoxin, and zearalenone) were added to the drinking water of mice to determine whether they were consumed or refused. Of the 10, only the trichothecenes--diacetoxyscirpenol, T-2 toxin, and vomitoxin--were refused. Refusal of 2 mg of the trichothecenes per liter was not enhanced by adding 100 mg of zearalenone per liter.     

Correlation of Biological to Chromatographic Data for Two Mycotoxins Elaborated by Fusarium

Thirty-seven identified strains of Fusarium, most of them isolated from fescue grass, were tested for their ability to elaborate mycotoxins in laboratory culture. The presence of the toxins was determined by infrared light, thin-layer chromatography, mouse toxicity, fungistatic effects, and phytotoxic properties. A good correlation was demonstrated between T-2 toxin detection by thin-layer chromatography and inhibition of Rhodotorula rubra by culture extracts. All of the strains producing either butenolide or T-2 toxin were toxic to mice with but one exception; those producing T-2 toxin inhibited growth of the yeast.     

Comparative yields of T-2 toxin and related trichothecenes from five toxicologically important strains of Fusarium sporotrichioides.

The range and comparative yields of T-2 toxin and related trichothecenes from five toxicologically important strains of Fusarium sporotrichioides, i.e., NRRL 3299, NRRL 3510, M-1-1, HPB 071178-13, and F-38, were determined. Lyophilized cultures of the five strains maintained in the International Toxic Fusarium Reference Collection were used to inoculate autoclaved corn kernels. Corn cultures were incubated at 15 degrees C for 21 days and analyzed for trichothecenes by thin-layer chromatography and capillary gas chromatography. All five strains produced T-2 toxin, HT-2 toxin, T-2 triol, and neosolaniol. Two strains also produced T-2 tetraol, and two others produced diacetoxyscirpenol. The highest producer of T-2 toxin (1,300 mg/kg), HT-2 toxin (200 mg/kg), T-2 triol (1.9 mg/kg), and neosolaniol (170 mg/kg) was NRRL 3510, which was originally isolated from millet associated with outbreaks of alimentary toxic aleukia in the USSR. The second highest producer of T-2 toxin (930 mg/kg) was NRRL 3299. The other three strains produced T-2 toxin at levels ranging from 130 to 660 mg/kg. Thus, the five strains differed considerably in the amounts of T-2 toxin and other trichothecenes produced under identical laboratory conditions. These strains are being maintained under optimal conditions for the preservation of Fusarium cultures and are available from the Fusarium Research Center, The Pennsylvania State University, University Park.     

Comparative yields of T-2 toxin and related trichothecenes from five toxicologically important strains of Fusarium sporotrichioides

The range and comparative yields of T-2 toxin and related trichothecenes from five toxicologically important strains of Fusarium sporotrichioides, i.e., NRRL 3299, NRRL 3510, M-1-1, HPB 071178-13, and F-38, were determined. Lyophilized cultures of the five strains maintained in the International Toxic Fusarium Reference Collection were used to inoculate autoclaved corn kernels. Corn cultures were incubated at 15 degrees C for 21 days and analyzed for trichothecenes by thin-layer chromatography and capillary gas chromatography. All five strains produced T-2 toxin, HT-2 toxin, T-2 triol, and neosolaniol. Two strains also produced T-2 tetraol, and two others produced diacetoxyscirpenol. The highest producer of T-2 toxin (1,300 mg/kg), HT-2 toxin (200 mg/kg), T-2 triol (1.9 mg/kg), and neosolaniol (170 mg/kg) was NRRL 3510, which was originally isolated from millet associated with outbreaks of alimentary toxic aleukia in the USSR. The second highest producer of T-2 toxin (930 mg/kg) was NRRL 3299. The other three strains produced T-2 toxin at levels ranging from 130 to 660 mg/kg. Thus, the five strains differed considerably in the amounts of T-2 toxin and other trichothecenes produced under identical laboratory conditions. These strains are being maintained under optimal conditions for the preservation of Fusarium cultures and are available from the Fusarium Research Center, The Pennsylvania State University, University Park.     

Aflatoxin B1 Uptake by Flavobacterium aurantiacum and Resulting Toxic Effects

Removal of aflatoxin B(1) from liquid cultures by resting and growing cells of Flavobacterium aurantiacum NRRL B-184 was studied. Spectrophotometic and thin-layer techniques served as aflatoxin assays. Cells grown in the presence of 5 ppm or higher levels of aflatoxin developed aberrant morphological forms. These toxin concentrations partially inhibited growth, and the nature of the inhibition suggested that aflatoxin interfered with cell wall synthesis. Incubation of 1.0 x 10(11) resting cells per milliliter with 7.0 mug/ml of aflatoxin B(1) during a 4-hr period facilitated complete toxin removal from a buffered aqueous medium. Autoclaved cells and cell wall preparations could remove a fraction of the aflatoxin of a test system. However, the toxin removed by autoclaved cells and cell walls could be extracted by washing with water but the aflatoxin B(1) removed by intact cells could not be extracted into the liquid phase. The uptake of aflatoxin B(1) by resting cells was sensitive to temperature and pH. Ruptured preparations of F. aurantiacum were not able to remove or modify the aflatoxin in an aqueous solution.     

Egg Production, Shell Thickness, and Other Physiological Parameters of Laying Hens Affected by T-2 Toxin

T-2 toxin has been reported to cause severe oral lesions and neural disturbances in young broiler chickens. T-2 toxin, when added at a level of 20 mug per g of feed, caused oral lesions but no abnormal neural disturbances in young broiler chickens. T-2 toxin, when added at a level of 20 mug per g of feed, caused oral lesions but no abnormal neural symptoms in laying hens. T-2 toxin had no effect on either hemoglobin, hematocrit values, erythrocyte count, plasma glucose, prothrombin times, or the sizes of the liver, spleen, pancreas, and heart. Lipid content of the liver was not altered. Feed consumption, however, was reduced, as were the total plasma protein and lipid concentrations and the total leukocyte count. Most important economically was the lowered egg production and a thinner egg shell. The timing and severity of the symptoms suggest that T-2 toxin causes primary oral lesions that reduce feed consumption with a consequent reduction in serum proteins and lipids, which culminate in decreased egg production. The leucopenia and thinner egg shell may be independent systemic effects of T-2 toxin in laying hens.     

Brine Shrimp (Artemia salina L.) Larvae as a Screening System for Fungal Toxins

Concentrations resulting in 50% mortality, determined with brine shrimp (Artemia salina L.) larvae exposed to known mycotoxins for 16 hr, were (mug/ml): aflatoxin G(1), 1.3; diacetoxyscirpenol, 0.47; gliotoxin, 3.5; ochratoxin A, 10.1; and sterigmatocystin, 0.54. 4-Acetamido-4-hydroxy-2-butenoic acid gamma-lactone gave no mortality at 10 mug/ml. Used as a screening system involving discs saturated with solutions of known mycotoxins, the larvae were relatively sensitive to aflatoxin B(1), diacetoxyscirpenol, gliotoxin, kojic acid, ochratoxin A, rubratoxin B, sterigmatocystin, stemphone, and T-2 toxin. Quantities of 0.2 to 2 mug/disc caused detectable mortality. The larvae were only moderately sensitive to citrinin, patulin, penicillic acid, and zearalenone which were detectable at 10 to 20 mug/disc. They were relatively insensitive to griseofulvin, luteoskyrin, oxalic acid, and beta-nitropropionic acid. The disc screening method indicated that 27 out of 70 fungal isolates from foods and feeds grown in liquid or solid media produced chloroform-extractable toxic material. Examination of toxic extracts by thin-layer chromatography for 17 known mycotoxins showed that the toxicity of eight isolates could be attributed to aflatoxin B(1) and B(2), kojic acid, zearalenone, T-2 toxin, or ochratoxin A. Nine out of 32 of these fungal isolates grown in four liquid media yielded toxic culture filtrates from at least one medium. Chemical tests for kojic, oxalic, and beta-nitropropionic acids showed the presence of one or two of these compounds in filtrates of seven of these nine isolates.     

Optimal Combination and Concentration of Antibiotics in Media for Isolation of Pathogenic Fungi and Nocardia asteroides

Strains of Blastomyces dermatitidis, Sporothrix schenckii, Histoplasma capsulatum, Cryptococcus neoformans, Nocardia asteroides, and Coccidioides immitis were tested for in vitro susceptibility to polymyxin, gentamicin, kanamycin, chloramphenicol, and neomycin at concentrations of 1, 2, 4, 8, and 16 mug/ml. Polymyxin was the most inhibitory and gentamicin was the least inhibitory of the five antibiotics. Two Histoplasma mycelial strains were partially inhibited by 2 and 8 mug of gentamicin per ml and showed at least a 2+ growth at the higher antibiotic concentration. Kanamycin and neomycin produced significant inhibition of N. asteroides but otherwise were noninhibitory. A combination of chloramphenicol and kanamycin, each at 16 mug/ml, and gentamicin, at 4 mug/ml, was noninhibitory to the strains tested except for N. asteroides. Chloramphenicol at 16 mug/ml was not inhibitory for N. asteroides. The results suggest that the optimal antibiotic combination to use in the isolation of fungi and higher bacteria is chloramphenicol, 16 mug/ml, and gentamicin, 4 mug/ml. Addition of sheep blood (5%) had no effect on antibiotic susceptibility of the organisms studied.     

Trichothecene Production in Liquid Stationary Cultures of Fusarium tricinctum NRRL 3299 (Synonym: F. sporotrichioides): Comparison of Quantitative Brine Shrimp Assay with Physicochemical Analysis

Stationary liquid cultures of Fusarium tricinctum NRRL 3299 (synonym: F. sporotrichioides) produce T-2 toxin, neosolaniol, diacetoxyscirpenol, and HT-2 toxin when cultured on peptone-enriched Czapek Dox medium. At 15 and 27°C, maximum T-2 toxin yield (265 and 50 μg/ml) was found after 10 to 14 and 7 days, respectively. The T-2 toxin in the culture medium was metabolized rapidly at 27°C and slowly at 15°C. Addition of 0.025% (wt/vol) sorbic acid to the medium resulted in an increased production of trichothecenes at 15°C (400 μg of T-2 per ml after 14 days). Trichothecenes in the culture liquid were determined by the brine shrimp bioassay and physicochemical analysis. The brine shrimp assay was improved by using modern bioassay equipment, including tissue culture trays and multipipettes, and by a standardized approach with positive and negative controls. The physicochemical analysis was based on adsorption of the trichothecenes onto Amberlite XAD-2 columns, derivatization with trifluoroacetic anhydride followed by capillary gas chromatography, and identification by mass spectrometry (as many as 17 trichothecenes were detected in the culture medium). The brine shrimp assay offers an interesting monitoring system for the quantitation of T-2 toxin and should be useful for studies on production of this toxin in culture. Specific information on less toxic trichothecenes, however, requires a more time-consuming chemical analysis.     

Role of pyrimidine dimer excision in loss of potential streptomycin resistance mutations of ultraviolet-irradiated Escherichia coli on phosphate-buffered agar.

The frequency of ultraviolet (UV)-induced mutations to streptomycin resistance dropped rapidly when starved Escherichia coli strains WP-2 B/r and B/r T- were incubated on phosphate-buffered agar (PBA), but was reduced only slightly in a WP-2 hcr- mutant. During postirradiation, incubation viability remained approximately constant. Cells given an optimal recovery treatment with photo-reactivating light showed no further recovery if subsequently incubated on PBA. At least 70% of the mutations induced to streptomycin resistance by UV could be repaired. The loss of potential streptomycin-resistant mutants was markedly reduced in strain B/r T- when 5 mug of acriflavin or 700 mug of caffeine per ml was added to PBA. The excision of UV-induced thymine-containing dimers from E. coli tb/r T- was investigated. Dimer excision progressed more slowly when the cells were incubated on PBA containing acriflavin or caffeine. There was no congruity between the kinetics of dimer excision and the kinetics of mutant loss. Our results indicate that removal of potential streptomycin-resistant mutants is considerably faster than the excision of pyrimidine dimers.     

Production and characterization of antibodies against HT-2 toxin and T-2 tetraol tetraacetate.

Three new immunogens which were prepared by conjugation of the carboxymethyl oxime (CMO) derivatives of HT-2 toxin, T-2 tetraol (T-2 4ol), and T-2 tetraol tetraacetate (T-2 4Ac) to bovine serum albumin (BSA) were tested for the production of antibodies against the major metabolites of T-2 toxin. Antibodies against HT-2 toxin and T-2 4Ac were obtained from rabbits 5 to 10 weeks after immunizing the animals with CMO-HT-2-BSA and CMO-T-2 4Ac-BSA conjugates. Immunization with CMO-T-2 4ol-BSA resulted in no antibody against T-2 4ol. The antibody produced against HT-2 toxin had great affinity for HT-2 toxin as well as good cross-reactivity with T-2 toxin. The relative cross-reactivities of anti-HT-2 toxin antibody with HT-2 toxin, T-2 toxin, iso-T-2 toxin, acetyl-T-2 toxin, 3'-OH HT-2, 3'-OH T-2, T-2 triol, and 3'-OH acetyl-T-2, were 100, 25, 10, 3.3, 0.25, 0.15, 0.12 and 0.08%, respectively. Antibody against CMO-T-2 4Ac was very specific for T-2 4Ac and had less than 0.1% cross-reactivity with T-2 toxin, HT-2 toxin, acetyl-T-2 toxin, diacetoxyscirpenol, deoxynivalenol, and deoxynivalenol triacetate as compared with T-2 4Ac. The detection limits for HT-2 toxin and T-2 4ol by radioimmunoassay were approximately 0.1 and 0.5 ng per assay, respectively.     

Application of an enzyme-linked immunosorbent assay for screening of T-2 toxin-producing Fusarium spp

Culture filtrates of Fusarium species were subjected without clean-up procedures to an indirect competitive enzyme-linked immunosorbent assay with anti-T-2 toxin monoclonal antibody. Fusarium sporotrichioides, F. poae, F. tricinctum, and F. culmorum strains were positive for T-2 toxin, with a minimum detection limit of 5 pg per assay (100 pg/ml of culture filtrate), and the assay data correlated well with the gas-liquid chromatographic data.     

Application of an enzyme-linked immunosorbent assay for screening of T-2 toxin-producing Fusarium spp.

Culture filtrates of Fusarium species were subjected without clean-up procedures to an indirect competitive enzyme-linked immunosorbent assay with anti-T-2 toxin monoclonal antibody. Fusarium sporotrichioides, F. poae, F. tricinctum, and F. culmorum strains were positive for T-2 toxin, with a minimum detection limit of 5 pg per assay (100 pg/ml of culture filtrate), and the assay data correlated well with the gas-liquid chromatographic data.     

Production and characterization of antibodies against HT-2 toxin and T-2 tetraol tetraacetate

Three new immunogens which were prepared by conjugation of the carboxymethyl oxime (CMO) derivatives of HT-2 toxin, T-2 tetraol (T-2 4ol), and T-2 tetraol tetraacetate (T-2 4Ac) to bovine serum albumin (BSA) were tested for the production of antibodies against the major metabolites of T-2 toxin. Antibodies against HT-2 toxin and T-2 4Ac were obtained from rabbits 5 to 10 weeks after immunizing the animals with CMO-HT-2-BSA and CMO-T-2 4Ac-BSA conjugates. Immunization with CMO-T-2 4ol-BSA resulted in no antibody against T-2 4ol. The antibody produced against HT-2 toxin had great affinity for HT-2 toxin as well as good cross-reactivity with T-2 toxin. The relative cross-reactivities of anti-HT-2 toxin antibody with HT-2 toxin, T-2 toxin, iso-T-2 toxin, acetyl-T-2 toxin, 3'-OH HT-2, 3'-OH T-2, T-2 triol, and 3'-OH acetyl-T-2, were 100, 25, 10, 3.3, 0.25, 0.15, 0.12 and 0.08%, respectively. Antibody against CMO-T-2 4Ac was very specific for T-2 4Ac and had less than 0.1% cross-reactivity with T-2 toxin, HT-2 toxin, acetyl-T-2 toxin, diacetoxyscirpenol, deoxynivalenol, and deoxynivalenol triacetate as compared with T-2 4Ac. The detection limits for HT-2 toxin and T-2 4ol by radioimmunoassay were approximately 0.1 and 0.5 ng per assay, respectively.     

Effect of Iron and Salt on Prodigiosin Synthesis in Serratia marcescens

Serratia marcescens wild-types ATCC 264 and Nima grew but did not synthesize prodigiosin in a glycerol-alanine medium containing 10 ng of Fe per ml. Wild-type 264 required the addition of 0.2 mug of Fe per ml for maximal growth and prodigiosin synthesis; Nima required 0.5 mug of Fe per ml. Three percent, but not 0.1%, sea salts inhibited prodigiosin synthesis in a complex medium containing up to 10 mug of Fe per ml. NaCl was the inhibitory sea salt component. The inhibition was not specific for NaCl; equimolar concentrations of Na(2)SO(4), KCl, and K(2)SO(4) also inhibited prodigiosin synthesis. Experiments with strains 264 and Nima and with mutant WF which cannot synthesize 4-methoxy-2-2'-bipyrrole-5-carboxyaldehyde (MBC), the bipyrrole moiety of prodigiosin, and with mutant 9-3-3 which cannot synthesize the monopyrrole moiety 2-methyl-3-amylpyrrole (MAP) showed that both MBC synthesis and the reaction condensing MAP and MBC to form prodigiosin were relatively more sensitive to NaCl inhibition than the MAP-synthesizing step. The capacity of whole cells to condense MAP and MBC was present, but inactive, in cells grown in NaCl; removal of the NaCl from non-proliferating salt-grown cells restored the activity. Other evidence suggests the existence of a common precursor to the MAP- and MBC-synthesizing pathways.     

Effect of Inoculum Size on In Vitro Susceptibility Testing with Lincomycin

There is disagreement in the literature as to whether lincomycin is primarily a bacteriostatic or a bactericidal agent against gram-positive cocci and also regarding the levels of activity of this agent against susceptible microorganisms. These questions were examined in a study of the effect of inoculum size on the results of tube dilution susceptibility determinations with lincomycin against 49 clinical isolates of Staphylococcus aureus and 25 strains of streptococci and pneumococci. Lincomycin was both highly active and bactericidal when tested against 40 strains of S. aureus with inocula containing a maximum of 10(4) cells per ml [median minimal inhibitory concentration (MIC), 0.78 mug/ml; median minimal bactericidal concentration (MBC), 1.56 mug/ml]. With inocula of 10(5) cells per ml, lincomycin was primarily bacteriostatic (median MIC, 1.56 mug/ml; median MBC, 12.5 mug/ml). There were further decreases in inhibitory levels and significant losses of bactericidal activity when inocula containing more than 10(7) cells were tested (median MIC, 3.13 mug/ml; median MBC > 100 mug/ml). Similar measurements with streptococci and pneumococci revealed a lesser effect of inoculum size. The mean MBC value for alpha-hemolytic streptococci increased from 0.40 to 1.05 mug/ml with an increase in inocula from 10(4) to 10(6) cells per ml, but without a marked increase in MIC values. Similar results were obtained for beta-hemolytic streptococci and pneumococci.     

Production of antibody against T-2 toxin.

Antibody against T-2 toxin was obtained after immunization of rabbits with bovine serum albumin-T-2 hemisuccinate conjugate. The antibody had greatest binding efficiency for T-2 toxin, less efficiency for HT-2, and least for T-2 triol. Cross-reaction of antibody with neosolaniol, T-2 tetraol, and 8-acetyl-neosolaniol was very weak. Diacetoxyscirpenol, trichodermin, vomitoxin, and verrucarin A essentially gave no cross-reaction with the antibody. The sensitivity of the binding assay for T-2 toxin detection was in the range of 1 to 20 ng per assay. Detailed methods for the preparation of the conjugate and the production of immune serum and methods for antibody determination are described.     

丁烯羟酸内酯对公鸡花种子萌发和赤霉素含量的影响

从植物燃烟中提取的丁烯羟酸内酯（buten01ide）能提高公鸡花种子的萌发率,并促进其萌发后的生长。光可以增强丁烯羟酸内酯的作用。公鸡花对外源GA的响应与对丁烯羟酸内酯的响应类似,二者有协同效应。酶联免疫法测定结果显示,在种子尚未萌发时,经丁烯羟酸内酯处理的种子中内源赤霉素含量低于不做处理的;萌发生长后,其赤霉素含量则高于不做处理的。因此认为,丁烯羟酸内酯可能是通过增加种子对GA的敏感性而起作用的。     

Neural Disturbances in Chickens Caused by Dietary T-2 Toxin

Graded concentrations of dietary T-2 toxin (0, 1, 2, 4, 8, and 16 mug/g) were fed to groups of 40 chickens. T-2 toxin was found to cause an abnormal positioning of the wings, hysteroid seizures, and impaired righting reflex in young chickens. The abnormal wing positioning occurred spontaneously or as the result of dropping from a height of 1 meter. The seizures could be elicited by rough handling or loud noises. The seizures and the abnormal wing posture would not occur again when the stimulus was repeated unless a rest period of 3 to 6 h was allowed. The loss of righting reflex could be demonstrated at any time. The total incidence of neural symptoms was dependent on the length of exposure to T-2 toxin and to its concentration. Neural toxicity occurred at dosages of 4, 8, and 16 mug per g of diet, which are the same doses that retard growth. This neural toxicity of T-2 toxin in chickens is similar to the neural disturbances associated with alimentary toxic aleukia, a nutritional toxicosis of humans produced by eating moldy grain. T-2 toxin has been implicated also in moldy corn toxicosis which has neural manifestations in horses and swine.