Mycotoxin profile of Fusarium langsethiae isolated from wheat in Italy: production of type‐A trichothecenes and relevant glucosyl derivatives

Fusarium langsethiae, formally described as a new species over a decade ago, has been identified as the main producer of HT‐2 (HT2) and T‐2 (T2) toxins in Europe in small cereal grains. Mycotoxin contamination caused by this Fusarium species can represent a food safety hazard that deserves further attention. In the present work, the mycotoxin profile in wheat cultures of F. langsethiae is presented with particular reference to the production of major type‐A trichothecenes and their glucosyl derivatives. F. langsethiae isolates, representative of the major Italian wheat cultivation areas, were tested for the production of T2, HT2, diacetoxyscirpenol (DAS) and neosolaniol (NEO), and relevant glucosyl derivatives. Liquid chromatography‐tandem mass spectrometry (LC‐MS/MS) was used for the identification and chemical characterization of these metabolites. F. langsethiae isolates under investigation resulted to be potent producers of T2, HT2 and NEO. Furthermore, a well‐defined set of isolates, all originating from Central Italy, produced also DAS. All isolates were found to be able to produce HT2 glucosyl derivatives, whereas only traces of T2 glucoside were detected in one sample. Furthermore, two mono‐glucosyl derivatives of NEO and one mono‐glucoside derivative of DAS were identified and characterized. The screening for the presence/absence of glucosylated trichothecenes in analyzed fungal extracts revealed a general co‐occurrence of these derivatives with the parent toxin at levels that could be roughly estimated to account up to 37% of the relevant unconjugated toxin. This is the first report of the production of glucosylated trichothecenes by F. langsethiae cultured on small grains. Copyright © 2013 John Wiley & Sons, Ltd.     

Experimental design for in-house validation of a screening immunoassay kit. The case of a multiplex dipstick for Fusarium mycotoxins in cereals

The benefits of using rapid qualitative methods to verify compliance of food and feed with legislation requirements include user-friendly format, the possibility of detection without expensive instrumentation, rapid response and affordable price. Prior to their use, however, the methods have to pass validation experiments, in order to assess their performance profile. An experimental protocol for in-house validation of a screening immunoassay has been designed and applied to evaluate performance characteristics of a multiplex dipstick kit for the determination of major Fusarium toxins, namely zearalenone, T-2 and HT-2 toxins, deoxynivalenol and fumonisins in wheat and maize. The test is intended for screening of cereals on the presence/absence of these mycotoxins at maximum permitted levels established by European legislation or target levels. The response of the measurement is determined with a reader device. Samples classified as negative are considered as compliant, whereas positive samples need to be re-analysed with confirmatory methods. The in-house validation design consisted of three steps, namely (1) estimating the precision of the method including "between day" effects and influences from different varieties of the matrices, (2) establishing robust cutoff values for the dipstick response at target mycotoxin levels assuming an acceptable rate of false negative results of 5 % and (3) assessment of the rate of false positive results of blank samples and samples containing the target analytes below the legal limits. The total precision expressed as relative standard deviation and determined individually for each analyte/concentration/matrix combination varied from 9 to 30 % and was considered as acceptable. In 17 out of 28 cases, the repeatability standard deviation was the most important factor. The predominance of the repeatability compared to the other factors (matrix, days) was an indicator for the ruggedness of the assay. The validation study demonstrated that the test was able to differentiate blank samples from samples contaminated at target mycotoxin levels with a false positive rate lower than 6 %. Considering realistic mycotoxin occurrence in European samples, significant economical benefits can be expected when using the test under real-world conditions.

Random replicators with high-order interactions

We use tools of the equilibrium statistical mechanics of disordered systems to study analytically the statistical properties of an ecosystem composed of N species interacting via random mutual interactions, as well as via deterministic self-interactions of order p>/=2. We show that the main effect of increasing the order of the interactions among the species is to make the system less competitive, in the sense that the fraction of extinct species is greatly reduced. In addition, we find that for p>2 there is a threshold value which gives a lower bound to the concentration of the surviving species, preventing then the existence of rare species and, consequently, increasing the robustness of the ecosystem to external perturbations.     

Convergence of a relaxation scheme for hyperbolic systems of conservation laws

Summary. This paper concerns the study of a relaxation scheme for hyperbolic systems of conservation laws. In particular, with the compensated compactness techniques, we prove a rigorous result of convergence of the approximate solutions toward an entropy solution of the equilibrium system, as the relaxation time and the mesh size tend to zero. Received September 29, 1998 / Revised version received December 20, 1999 / Published online August 24, 2000     

A ketogenic diet increases succinic dehydrogenase (SDH) activity and recovers age-related decrease in numeric density of SDH-positive mitochondria in cerebellar Purkinje cells of late-adult rats

Ketogenic diets (KDs) have been applied in the therapy of paediatric epilepsy for nearly a century. Recently, beneficial results have also been reported on metabolic disorders and neurodegeneration, designating aged individuals as possible recipients. However, KDs efficacy decrease after the suckling period, and very little is known about their impact on the aging brain. In the present study, the effect on the neuronal energetic supply of a KD containing 20% of medium chain triglycerides (MCT) was investigated in Purkinje cells of the cerebellar vermis of late-adult (19-month-old) rats. The animals were fed with the KD for 8 weeks, and succinic dehydrogenase (SDH) activity was cytochemically determined. The following parameters of SDH-positive mitochondria were evaluated by the use of a computer-assisted image analysis system connected to a transmission electron microscope: numeric density (Nv), average volume (V), volume density (Vv), and cytochemical precipitate area/mitochondrial area (R). Young, age-matched, and old animals fed with a standard chow were used as controls. We found significantly higher Nv in MCT-KD-fed rats vs. all the control groups, in young vs. late-adult and old controls, and in late-adult vs. old controls. V and Vv showed no significant differences among the groups. R was significantly higher in MCT-KD-fed rats vs. all the control animals, and in old vs. young and late-adult controls. Present data indicate that the ketogenic treatment counteracted age-related decrease in numeric density of SDH-positive mitochondria, and enhanced their metabolic efficiency. Given the central role of mitochondrial impairment in age-related physio-pathological changes of the brain, these findings may represent a starting point to examine novel potentialities for KDs.     

Stable carbon and nitrogen isotope discrimination and turnover in a small-bodied insectivorous lizard

Laboratory experiments are useful for estimating the carbon and nitrogen isotope discrimination factors and turnover rates that are critical for drawing field-based inferences on consumer diets using stable isotopes. Although the utility of these discrimination factors is widely recognized, work in terrestrial systems has largely been limited to studies involving mammals and birds. In contrast, scant attention has been paid to the application of isotopic techniques to reptiles, despite their broad diversity in terms of numbers of species as well as their trophic roles. Here we estimate carbon and nitrogen isotope discrimination factors and turnover for the tree lizard (Urosaurus ornatus) using a diet-switch experiment. Lizards were collected from a C₄-dominated grassland and then switched to C₃-based diet (crickets) in the laboratory. We estimated discrimination by lizard claw tissue as Δ¹³C?=?1.2?±?0.1?‰ for carbon and Δ¹⁵N?=?0.7?±?0.1?‰ (mean?±?1 SE) for nitrogen, with 95?% turnover occurring after ～15.5 days. These estimates should be appropriate for use in trophic studies of U. ornatus, and possibly other related small-bodied insectivorous lizards.     

Analysis of T-2 and HT-2 toxins in cereal grains by immunoaffinity clean-up and liquid chromatography with fluorescence detection

A sensitive, precise and accurate method has been developed for the simultaneous determination of T-2 and HT-2 toxins in cereal grains at ppb levels using high-performance liquid chromatography (HPLC) with fluorescence detection and 1-antroylnitrile (1-AN) as labeling reagent after immunoaffinity clean-up. Cereal samples were extracted with methanol/water (90:10, v/v), and the extracts were cleaned-up through commercially available immunoaffinity columns containing monoclonal anti-T-2 antibodies (T-2 test HPLC, Vicam). T-2 and HT-2 toxins were quantified by reversed-phase HPLC with fluorometric detection (excitation wavelength 381 nm, emission wavelength 470 nm) after derivatization with 1-AN. The monoclonal antibody showed 100% cross-reactivity with both T-2 and HT-2 toxin, and the immunoaffinity column clean-up was effective up to 1.4 microg of both toxins. The method was successfully applied to the analysis of T-2 and HT-2 toxins in wheat, maize and barley. Recoveries from spiked samples with toxin levels from 25 to 500 microg/kg ranged from 70% to 100%, with relative standard deviation generally lower than 8%. The limit of detection of the method was 5 microg/kg for T-2 toxin and 3 microg/kg for HT-2 toxin, based on a signal-to-noise ratio 3:1. HT-2 toxin was detected in ten naturally contaminated wheat samples out of 14 samples analyzed, with toxin levels ranging from 10 to 71 microg/kg; three of them contained also T-2 toxin up to 12 microg/kg.     

Shock Layers Interactions for a Relaxation Approximation to Conservation Laws

We consider the interaction between shock waves in a semilinear relaxation approximation to N×N systems of conservation laws. This interaction is described by proving the existence of a particular solution to the relaxing system, with a certain asymptotic as Received July 1998     

Simultaneous LC–MS/MS determination of aflatoxin M<Subscript>1</Subscript>, ochratoxin A,deoxynivalenol, de-epoxydeoxynivalenol, α and β-zearalenols and fumonisin B<Subscript>1</Subscript> in urine as a multi-biomarker method to assess exposure to mycotoxins

Humans and animals can be simultaneously exposed through the diet to different mycotoxins, including aflatoxins, ochratoxin A, deoxynivalenol, zearalenone, and fumonisins, which are the most important. Evaluation of the frequency and levels of human and animal exposure to these mycotoxins can be performed by measuring the levels of the relevant biomarkers in urine. Available data on the toxicokinetics of these mycotoxins in animals suggest that aflatoxin M₁ (AFM₁), ochratoxin A (OTA), deoxynivalenol (DON)/de-epoxydeoxynivalenol (DOM-1), alpha-zearalenol (α-ZOL)/beta-zearalenol (β-ZOL), and fumonisin B₁ (FB₁) can be used as urinary biomarkers. A liquid chromatographic–tandem mass spectrometric method has been developed for simultaneous determination of these mycotoxin biomarkers in human or animal urine. Urine samples were purified and concentrated by a double cleanup approach, using a multitoxin immunoaffinity column and a reversed-phase SPE Oasis HLB column. Separation of the biomarkers was performed by reversed-phase chromatography using a multi-step linear methanol–water gradient containing 0.5% acetic acid as mobile phase. Detection and quantification of the biomarkers were performed by triple quadrupole mass spectrometry (LC–ESI-MS/MS). The clean-up conditions were optimised to obtain maximum analyte recovery and high sensitivity. Recovery from spiked samples was performed at four levels in the range 0.03–12 ng mL⁻¹, using matrix-matched calibration curves for quantification. Mean recoveries of the biomarkers tested ranged from 62 to 96% with relative standard deviations of 3–20%. Enzymatic digestion with β-glucuronidase/sulfatase resulted in increased concentrations of the biomarkers, in both human and pig urine, in most samples containing measurable concentrations of DON, DOM-1, OTA, α-ZOL, or β-ZOL. A highly variable increase was observed between individuals. Co-occurrence of OTA and DON in human urine is reported herein for the first time.     

Identification and characterization of new Fusarium masked mycotoxins,T2 and HT2 glycosyl derivatives,in naturally contaminated wheat and oats by liquid chromatography–high‐resolution mass spectrometry

The presence of glucoside derivatives of T‐2 and HT‐2 toxins (type A trichothecene mycotoxins) in naturally contaminated wheat and oats is reported for the first time. The use of advanced high‐resolution mass spectrometry based on Orbitrap technology allowed to obtain molecular structure details by measuring exact masses of main characteristic fragments, with mass accuracy lower than 2.8 ppm (absolute value). A monoglucoside derivative of T‐2 toxin and two monoglucoside derivatives of HT‐2 toxin were identified and characterized. The analysis of their fragmentation patterns provided evidence for glucosylation at C‐3 position for T‐2 toxin and at C‐3 or C‐4 position for HT‐2 toxin. A screening for the presence of these new masked forms of mycotoxins was carried out on a set of naturally contaminated wheat and oats samples. On the basis of peak area ratio between glucoside derivatives and free T‐2 and HT‐2 toxins, the presence of glucoside derivatives was more likely in wheat than in oats samples. The present work confirms the widespread occurrence of trichothecene glucosides in cereal grains naturally contaminated with the relevant unconjugated toxins, thus suggesting the importance of developing suitable analytical methods for their detection. Besides toxicity studies, tracking down these new masked forms of trichothecenes along the food/feed chain would enable to collect information on their relevance in human/animal exposure to mycotoxin risk. Copyright © 2012 John Wiley & Sons, Ltd.