Mycotoxin occurrence in commodities, feeds and feed ingredients sourced in the Middle East and Africa.

Between February and October 2009, 324 grain, feed and feed commodity samples were sourced directly at animal farms or feed production sites in Middle East and Africa and tested for the presence of A- and B-trichothecenes, zearalenone, fumonisins, aflatoxins and ochratoxin A, or for selected groups of mycotoxins only. Samples were analyzed after clean-up by immunoaffinity or solid-phase extraction followed by HPLC with derivatization where appropriate and fluorescence, UV or mass spectrometric detection. The percentage of positive samples of B-trichothecenes ranged from 0 to 87% of tested samples. The prevalence of fumonisins in the different countries was >50% in most cases. Zearalenone was present in tested commodities from all countries except three. The presence of aflatoxin in analyzed samples varied from 0 to 94%. Ochratoxin A was present in 67% of samples in Sudan and in 100% of Nigerian samples. No A-trichothecenes were found in this survey.

Rapid fluorometric test for the quantitative determination of deoxynivalenol in raw cereals.

Fast test systems fulfilling legislative specifications are required to determine mycotoxin contamination in unprocessed cerealse.g. at grain elevators, import and export terminals, or the milling and brewing industry to prevent contamination of food and feed.We describe the first tests of a novel fluorescence-based test for deoxynivalenol (DON), which will be commercially available within the next few months.The analytical procedure of the new test takes less than 15 minutes for extraction, purification, derivatization and measurement. The system's advantage is its speed and easy procedure providing quantitative DON determination.To ensure an international standard, the validation procedure for wheat, barley and maize will be performed following USDA/GIPSA requirements (03/2006) for DON tests.

Characterization and application of isotope-substituted (13C15)-deoxynivalenol (DON) as an internal standard for the determination of DON.

The powerful combination of liquid chromatography and mass spectrometry (MS) is often limited by matrix effects during ionization in the MS ion source. The use of fully isotope-substituted (13C15)-deoxynivalenol ((13C15)-DON) as an internal standard (IS) corrects matrix effects and improves the accuracy of analytical methods using mass spectrometry for the quantitative determination of the Fusarium mycotoxin deoxynivalenol (DON). The IS was characterized with respect to its chromatographic purity by liquid chromatography-ultraviolet light and its isotope distribution by time-of-flight mass spectrometry. Its low-energy collision-induced dissociation behaviour was compared with DON. Moreover, this work describes the successful application of (13C15)-DON as IS for the determination of DON in maize using high-performance liquid chromatography (HPLC) electrospray (ESI) with tandem mass spectrometry. The results demonstrate that the IS can successfully correct for fluctuations during extraction and clean-up of the sample as well as the ionization of DON in the MS ion source. Random variations in ionization affect the IS in the same way as the analyte. Recoveries for DON in maize of 76% +/- 1.9% (external calibration) or 101% +/- 2.4% (internal calibration) were reached, respectively, after sample clean-up.

Performance of new clean-up column for the determination of ochratoxin A in cereals and foodstuffs by HPLC-FLD.

The performance of the newly developed Mycosep 229 Ochra and Multisep 229 Ochra clean-up columns for ochratoxin A (OTA) determination was evaluated. OTA was subsequently analysed using RP-HPLC with fluorescence detection. Recoveries for frequently contaminated commodities, like cereals, red wine, raisins and green coffee, were estimated. The recoveries obtained for the Mycosep 229 Ochra column were in the range from 87.9 +/- 12.5% (n = 6) for wheat to 99.4 +/- 2.7% (n = 24) for raisins. For Multisep 229 Ochra, recoveries from 76.5 +/- 8.0% (n = 6) for barley to 86.4 +/- 1.4% (n = 24) for raisins were achieved. Limits of detection for all matrices investigated (maize, wheat, rice, barley, raisins, green coffee beans, red wine) were in the range 0.4-2.4 microg kg(-1). The trueness of the method was tested using a certified reference material.

Type A-trichothecenes - Quantitative analysis using LC-MS and occurrence in Austrian maize and oats.

During this study a method was developed for the quantitative determination of diacetoxyscirpenol (DAS), T-2 toxin and HT-2 toxin using deuterated T-2 toxin (T-2 d3 toxin) as an internal standard. The described method involves a clean up step of maize extract by the use of Mycosep® 227 columns a chromatographic separation on a Zorbax® bonus-RP-column (2,1×150mm) and the detection and quantification step on a mass spectrometer in SIM (Selected Ion Monitoring) mode.Data on the occurrence of three type A trichothecenes in Austrian maize, maize silage and oats were collected. It could be shown that maize and silage samples harvested in 2002 were only contaminated to a small extent with T-2 toxin (8% of the maize, 0% of the silage) and with HT-2 toxin (30% of the maize, 18% of the silage). But most of the analysed oats samples showed significant levels of T-2 toxin (64%) or HT-2 toxin (82%).

Toxicity of ochratoxin A in aBrevibacillus brevis - Growth inhibition assay.

Ochratoxin A (OTA) is a nephrotoxic, carcinogenic and immunosuppressive mycotoxin. It can be detoxified by various microorganisms, e.g. different yeast strains, via metabolisation into ochratoxin α (OTα). Within this study a growth inhibition assay was developed to compare the toxicity of OTA and its degradation product OTα. As an indicator organismBrevibacillus brevis was used. The assay was performed in microtiterplates. Growth inhibition was determined by comparing the optical density values ofBrevibacillus brevis cultures grown in medium supplemented with OTA/OTα and OTA/OTα-free medium, respectively.It could be shown thatB. brevis is sensitive to OTA (EC100=0.5 mg/L±0.03 mg/L), which is not the case for its metabolite OTα. Therefore this bioassay is a useful tool to show the detoxification of OTA to OTα by microbial degradation.

Investigation of different yeast strains for the detoxification of ochratoxin A.

High concentrations of ochratoxin A (OTA) in feed lead to growth depression in animals. It has been reported that binders can be used for deactivating aflatoxins but not for other mycotoxins without negatively influencing the animals health. In this study a strain from the genus ofTrichosporon with the ability to cleave ochratoxin A very selectively into phenylalanine and the non-toxic ochratoxin α (OTα) could be isolated. This strain was selected from a pool of OTA detoxifying microorganism by carrying out several investigations.Trichosporon sp. nov. can be fermented and stabilized. In a feeding trial with broilers lyophilizedTrichosporon-cells could compensate performance losses caused by OTA.

Determination of fumonisins and hydrolyzed fumonisin B1 in microbial culture media by LC/ESI-MS.

In order to analyze liquid growth media for fumonisin B1, B2 and hydrolyzed fumonisin B1 (HFB1) after incubation with microorganisms for degradation studies, a liquid chromatography/electrospray ionisation mass spectrometry (LC/ESI-MS) method was developed. The use of phytosphingosine hydrochloride (PSH) and T-2-toxin-d3 (T2d3) as internal standard has been tested. The detection limit established with PSH as an internal standard was about 20 ng/mL for FB1 and FB2 and about 50 ng/mL for HFB1. The developed method allows the rapid, simultaneous and quantitative determination of these analytes in microbial culture media without any cleanup.

Structural characterization of metabolites after the microbial degradation of type A trichothecenes by the bacterial strain BBSH 797.

Contamination of feed with trichothecenes, a group of Fusarium mycotoxins, leads to losses in performance due to their immunosupressive effects and the negative effect on the gastrointestinal system in animal production. A possible way of detoxification is microbial degradation, which was the focus of this study. A bacterial strain--BBSH 797--which can degrade some mycotoxins of the trichothecene group, has already been isolated. It transforms deoxynivalenol (DON) into its metabolite DOM-1, the non-toxic deepoxide of DON. Analogous to the microbial degradation of DON, the transformation of six different type A trichothecenes was observed. The metabolites appearing were characterized by GC-MS after derivatization with TRI-SIL TBT. Two metabolites were additionally, identified by liquid chromatography-mass spectrometry with particle beam interface (LC-PB-MS) with electron impact (EI)-ionization mode. The major finding was that scirpentriol was completely transformed into its non-toxic metabolite deepoxy scirpentriol, while the mycotoxin T-2 triol underwent a more complicated metabolism. According to the study, T-2-triol was degraded into its non-toxic deepoxy form and into T-2 tetraol, which was then further metabolized to deepoxy T-2 tetraol. GC-MS after derivatization with TRI-SIL TBT was suitable for the structural characterization of trichothecenes and their degradation products. Besides the mass spectra of already known degradation products, spectra of new metabolites could be recorded by LC-PB-MS.

Feeding OTA to pigs: Analytical evaluation of a trial.

Methods were developed to analyse the concentrations of ochratoxin A (OTA) and its main metabolite ochratoxin α in blood plasma of pigs and in their kidneys. By the use of these methods blood and kidneys of pigs that were fed contaminated feedstuff containing 1500 µg/kg ochratoxin A for the whole trial period of 42 days were analysed.High levels of 1500 - 2000 µg/I OTA were found in the plasma, while the concentration of OTA in the kidneys did not exceed 250 µg/kg at the end of the trial. Neither in the plasma nor in the kidneys of the animals fed contaminated feedstuff ochratoxin a could be detected.

Evidence of ochratoxin A-detoxification activity of rumen fluid, intestinal fluid and soil samples as well as isolation of relevant microorganisms from these environments.

Dietary ochratoxin A (OTA) has a negative impact on performance of chickens and pigs. To avoid losses in animal production through intake of this mycotoxin and to prevent carry over to humans, strategies for counteracting have to be developed. In contrast to physical and chemical detoxification methods inactivation of ochratoxins by enzymatic reactions represent a very specific and gentle process. For the development of a new feed additive various environments have been screened for microorganisms with the capability of degrading or of cleaving the phenylalanine-moiety of ochratoxin A. Two OTA-degrading bacterial strains were isolated from rumen fluid and four pure cultures capable of cleaving ochratoxin A were obtained from pig intestine. The highest number of ochratoxin A degrading strains were found amongst aerobic bacteria which have mainly been isolated from soil.

A conserved subtilisin-like protein TgSUB1 in microneme organelles of Toxoplasma gondii.

Proteolytic processing plays a significant role in the process of invasion by the obligate intracellular parasite Toxoplasma gondii. We have cloned a gene, TgSUB1, encoding for a subtilisin-type serine protease found in T. gondii tachyzoites. TgSUB1 protein is homologous to other Apicomplexan and bacterial subtilisins and is processed within the secretory pathway of the parasite. Initial cleavage occurs in the endoplasmic reticulum, after which the protein is transported to micronemes, vesicles that secrete early during host cell invasion. Upon stimulation of microneme secretion, TgSUB1 is cleaved into smaller products that are secreted from the parasite. This secondary processing is inhibited by brefeldin A and serine protease inhibitors. TgSUB1 is a candidate processing enzyme for several microneme proteins cleaved within the secretory pathway or during invasion.