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A glycopeptide fraction (GPF) from internal organs of green sea urchins (Strongylocentrotus droebachiensis M&uuml, ller, Strongylocentrotidae) has been reported to be an effective bronchitis treatment. In this study, we evaluated the pharmacokinetic and tissue distribution of GPF, following single and repeated intranasal (i/n) administration over the course of seven days in rats. The method measuring lactate dehydrogenase as biomarker was used to analyse the plasma and tissue concentrations of GPF. GPF appears in the plasma 15 min after single i/n administration (100 &micro, g/kg) and reaches its maximum at 45 min. The area under the curve (AUC)0&ndash, 24 and Cmax were similar using both i/n and intravenous administration, while mean residence time (MRT) and T1/2 after i/n administration were significantly higher compared with intravenous (i/v) administration. The absolute bioavailability of GPF after i/n administration was 89%. The values of tissue availability (ft) provided evidence about the highest concentration of GPF in the nose mucosa (ft = 34.9), followed by spleen (ft = 4.1), adrenal glands (ft = 3.8), striated muscle (ft = 1.8), kidneys (ft = 0.5), and liver (ft = 0.3). After repeated dose administration, GPF exhibited significantly higher AUC0&ndash, 24 and MRT, indicating its accumulation in the plasma.

The downstream processing of enveloped virus-like particles is very challenging because of the biophysical and structural similarity between correctly assembled particles and contaminating vesicular particles present in the feedstock. We used hydroxyl-functionalized polymethacrylate monoliths, providing hydrophobic and electrostatic binding contributions, for the purification of HIV-1 gag virus-like particles. The clarified culture supernatant was conditioned with ammonium sulfate and after membrane filtration loaded onto a 1 mL monolith. The binding capacity was 2 × 1012 /mL monolith and was only limited by the pressure drop. By applying either a linear or a step gradient elution, to decrease the ammonium sulfate concentration, the majority of double-stranded DNA (88-90%) and host cell protein impurities (39-61%) could be removed while the particles could be separated into two fractions. Proteomic analysis and evaluation of the p24 concentration showed that one fraction contained majority of the HIV-1 gag and the other fraction was less contaminated with proteins originated from intracellular compartments. We were able to process up to 92 bed volumes of conditioned loading material within 3 h and eluted in average 7.3 × 1011 particles per particle fraction, which is equivalent to 730 vaccination doses of 1 × 109 particles.

Glycans play key roles in host-pathogen interactions; thus, knowing the N-glycomic repertoire of a pathogen can be helpful in deciphering its methods of establishing and sustaining a disease. Therefore, we sought to elucidate the glycomic potential of the facultative amoebal parasite Acanthamoeba. This is the first study of its asparagine-linked glycans, for which we applied biochemical tools and various approaches of mass spectrometry. An initial glycomic screen of eight strains from five genotypes of this human pathogen suggested, in addition to the common eukaryotic oligomannose structures, the presence of pentose and deoxyhexose residues on their N-glycans. A more detailed analysis was performed on the N-glycans of a genotype T11 strain (4RE); fractionation by HPLC and tandem mass spectrometric analyses indicated the presence of a novel mannosylfucosyl modification of the reducing terminal core as well as phosphorylation of mannose residues, methylation of hexose and various forms of pentosylation. The largest N-glycan in the 4RE strain contained two N-acetylhexosamine, thirteen hexose, one fucose, one methyl, and two pentose residues; however, in this and most other strains analyzed, glycans with compositions of Hex(8-9)HexNAc(2)Pnt(0-1) tended to dominate in terms of abundance. Although no correlation between pathogenicity and N-glycan structure can be proposed, highly unusual structures in this facultative parasite can be found which are potential virulence factors or therapeutic targets.

The modification of α1,6-linked fucose residues attached to the proximal (reducing-terminal) core N-acetylglucosamine residue of N-glycans by β1,4-linked galactose (“GalFuc” epitope) is a feature of a number of invertebrate species including the model nematode Caenorhabditis elegans. A pre-requisite for both core α1,6-fucosylation and β1,4-galactosylation is the presence of a nonreducing terminal N-acetylglucosamine; however, this residue is normally absent from the final glycan structure in invertebrates due to the action of specific hexosaminidases. Previously, we have identified two hexosaminidases (HEX-2 and HEX-3) in C. elegans, which process N-glycans. In the present study, we have prepared a hex-2;hex-3 double mutant, which possesses a radically altered N-glycomic profile. Whereas in the double mutant core α1,3-fucosylation of the proximal N-acetylglucosamine was abolished, the degree of galactosylation of core α1,6-fucose increased, and a novel Galα1,2Fucα1,3 moiety attached to the distal core N-acetylglucosamine residue was detected. Both galactosylated fucose moieties were also found in two parasitic nematodes, Ascaris suum and Oesophagostomum dentatum. As core modifications of N-glycans are known targets for fungal nematotoxic lectins, the sensitivity of the C. elegans double hexosaminidase mutant was assessed. Although this mutant displayed hypersensitivity to the GalFuc-binding lectin CGL2 and the N-acetylglucosamine-binding lectin XCL, the mutant was resistant to CCL2, which binds core α1,3-fucose. Thus, the use of C. elegans mutants aids the identification of novel N-glycan modifications and the definition of in vivo specificities of nematotoxic lectins with potential as anthelmintic agents.

Concentration profile of zearalenone (ZON) and its metabolites in plasma, urine and faeces samples of horses fed with Fusarium toxin-contaminated oats is described. In plasma, β-zearalenol (β-ZOL) was detected at high levels on day 10 of the study (3.21-6.24 μg/l). β-Zearalenol and α-zearalenol were the major metabolites in urine. Zearalenone, α-ZOL and β-ZOL were predominantly found in faeces. Zearalanone could also be detected in urine (1.34-5.79 μg/l) and faeces (1 μg/kg). The degree of glucuronidation was established in all sample types, approximately 100% in urine and plasma. Low per cent of glucuronidation (4-15%) was found in faeces samples. The results indicate the main conversion of ZON into β-ZOL in horse. This finding could explain why horse is not susceptible to ZON in comparison with swine which produce α-ZOL as a predominant metabolite.

Trichomonad species are widespread unicellular flagellated parasites of vertebrates which interact with their hosts through carbohydrate-lectin interactions. In the past, some data have been accumulated regarding their lipo(phospho)glycans, a major glycoconjugate on their cell surfaces; on the other hand, other than biosynthetic aspects, few details about their N-linked oligosaccharides are known. In this study, we present both mass spectrometric and high-performance liquid chromatography data about the N-glycans of different strains of Trichomonas vaginalis, a parasite of the human reproductive tract. The major structure in all strains examined is a truncated oligomannose form (Man(5)GlcNAc(2)) with α1,2-mannose residues, compatible with a previous bioinformatic examination of the glycogenomic potential of T. vaginalis. In addition, dependent on the strain, N-glycans modified by pentose residues, phosphate or phosphoethanolamine and terminal N-acetyllactosamine (Galβ1,4GlcNAc) units were found. The modification of N-glycans by N-acetyllactosamine in at least some strains is shared with the lipo(phospho)glycan and may represent a further interaction partner for host galectins, thereby playing a role in binding of the parasite to host epithelia. On the other hand, the variation in glycosylation between strains may be the result of genetic diversity within this species.

Aflatoxin B(1) is a common contaminant of poultry feeds in tropical and subtropical climates. Research during the last five decades has well established the negative effects of the mycotoxin on health of poultry. However, the last ten years of relevant data have accentuated the potential of low levels of aflatoxin B(1) to deteriorate broiler performance. In this regard, any attempt to establish a dose-effect relationship between aflatoxin B(1) level and broiler performance is also complicated due to differences in types of broilers and length of exposure to the mycotoxin in different studies. Contrary to the prevalent notion regarding literature saturation with respect to aflatoxicosis of chicken, many areas of aflatoxicosis still need to be explored. Literature regarding effects of the mycotoxin on the gastrointestinal tract in this regard is particular scanty and non-conclusive. In addition to these issues, the metabolism of aflatoxin B(1) and recently proposed hypotheses regarding biphasic effects of the mycotoxin in broilers are briefly discussed.

Planarial species are of especial interest to biologists due to the phenomenon of pluripotency and, in comparison to other developmental processes, it can be hypothesised that glycan–lectin interactions may play a role. In order to examine the N-glycans of one of these organisms, Dugesia japonica, peptide:N-glycosidase A was employed and the released glycans were subject to pyridylamination, HPLC and mass spectrometric analysis. A range of oligomannosidic glycans was observed with a trimethylated Man5GlcNAc2 structure being the dominant species. Three glycans were also observed to contain deoxyhexose; in particular, a glycan with the composition Hex4HexNAc2Fuc1Me2 was revealed by exoglycosidase digestion, in combination with MS/MS, to contain a galactosylated core α1,6-fucose residue, whereas this core modification was found to be capped with a methylhexose residue in the case of a Hex5HexNAc2Fuc1Me3 structure. This is the first report of these types of structures in a platyhelminth and indicates that the ‘GalFuc’ modification of N-glycans is not just restricted to molluscs and nematodes. Copyright © 2011 John Wiley & Sons, Ltd.

The paper presents a new sample clean-up method based on immuno-ultrafiltration for the analysis of ochratoxin A in cereals. In contrast to immunoaffinity chromatography, in immuno-ultrafiltration, the antibodies are used in non-immobilised form. Ochratoxin A was extracted with ACN/water (60/40, v/v), and the extract was loaded onto the ultrafiltration device. After a washing step with phosphate-buffered saline, containing 0.05% Tween 20, ochratoxin A was eluted with MeOH/acetic acid (99/1, v/v). The detection of ochratoxin A was carried out with high-performance liquid chromatography and a fluorescence detector coupled to an electrochemical cell (Coring cell). The electrochemical cell was used to eliminate matrix interferences by oxidising matrix compounds. The method was validated by repeatedly analysing spiked barley and rye samples as well as a certified wheat reference material. Recoveries and standard deviations (1 SD) were found to be 71 ± 9%, 77 ± 12% and 77 ± 8% in wheat, barley and rye, respectively. The limit of detection (S/N = 3) and limit of quantitation (S/N = 10) were determined to be 0.4 μg kg(-1) and 1 μg kg(-1). The analysis of the certified reference material resulted in ochratoxin A concentrations which were in the range assigned by the producer. Additionally, the effect of the electrochemical cell on other widely used clean-up techniques, namely the immunoaffinity clean-up and multifunctional columns (Mycosep #229), was evaluated. In all clean-up methods, an improvement of the chromatogram quality was registered.

Eighty-one rice samples were purchased from different markets in Vienna and were analysed for their aflatoxin content. The samples were extracted using methanol in water (80/20 v/v) followed by immunoaffinity clean up. The determination was carried out by HPLC–FLD coupled to a Kobracell. Different samples including basmati rice, whole grain rice, long grain rice, short grain rice as well as puffed rice were investigated. Moreover, conventionally and organically produced rice were compared. The results revealed that 24 out of 81 samples contained detectable amounts of aflatoxins. Aflatoxin B1 could be quantified in 15 samples and aflatoxin B2 in one sample. The contamination range was noted to be between 0.45 μg kg−1 and 9.86 μg kg−1 for aflatoxin B1 and 1.5 μg kg−1 for aflatoxin B2. Aflatoxins G1 and G2 were not detected in any sample. Three samples exceeded the maximum levels set in the European Union; having AFB1 concentrations of 2.16, 2.85 and 9.86 μg kg−1. In the three organic produced rice samples only traces of aflatoxins were found.

Galectin CGL2 from the ink cap mushroom Coprinopsis cinerea displays toxicity toward the model nematode Caenorhabditis elegans. A mutation in a putative glycosyltransferase-encoding gene resulted in a CGL2-resistant C. elegans strain characterized by N-glycans lacking the β1,4-galactoside linked to the α1,6-linked core fucose. Expression of the corresponding GALT-1 protein in insect cells was used to demonstrate a manganese-dependent galactosyltransferase activity. In vitro, the GALT-1 enzyme showed strong selectivity for acceptors with α1,6-linked N-glycan core fucosides and required Golgi- dependent modifications on the oligosaccharide antennae for optimal synthesis of the Gal-β1,4-fucose structure. Phylogenetic analysis of the GALT-1 protein sequence identified a novel glycosyltransferase family (GT92) with members widespread among eukarya but absent in mammals.

This paper presents a limited survey of aflatoxin M1 (AFM1) in Indonesian milk. AFM1 concentrations of 113 fresh milk samples, collected in 2006 from farms in five different areas of the Yogyakarta Province were analysed. The fresh milk samples were taken directly from dairy farms before pasteurisation. Enzyme-linked immunosorbent assay (ELISA) was used for the analysis of milk samples. Results show that in 48 samples (42.5%) the AFM1 concentrations were less than 5 ng/L and in 31 samples (27.4%) AFM1 was found between 5 and 10 ng/L. In 34 samples (30.1%) the concentrations were above 10 ng/L. None of contaminated samples exceeded the European Union regulation limits of 25 and 50 ng AFM1/L for infant and adult consumption, respectively. Since AFM1 is derived from aflatoxin B1 (AFB1) contained in cow feedstuffs, based on the contamination levels of AFM1 found in this study, the exposure of animals to AFB1 seems to be low.

Enveloped virus-like particles (VLPs) are increasingly used as vaccines and immunotherapeutics. Frequently, very time consuming density gradient centrifugation techniques are used for purification of VLPs. However, the progress towards optimized large-scale VLP production increased the demand for fast, cost efficient and scale able purification processes. We developed a chromatographic procedure for purification of HIV-1 gag VLPs produced in CHO cells. The clarified and filtered cell culture supernatant was directly processed on an anion-exchange monolith. The majority of host cell impurities passed through the column, whereas the VLPs were eluted by a linear or step salt gradient; the major fraction of DNA was eluted prior to VLPs and particles in the range of 100⿿200nm in diameter could be separated into two fractions. The earlier eluted fraction was enriched with extracellular particles associated to exosomes or microvesicles, whereas the late eluting fractions contained the majority of most pure HIV-1 gag VLPs. DNA content in the exosome-containing fraction could not be reduced by Benzonase treatment which indicated that the DNA was encapsulated. Many exosome markers were identified by proteomic analysis in this fraction. We present a laboratory method that could serve as a basis for rapid downstream processing of enveloped VLPs. Up to 2000 doses, each containing 1ÿ109 particles, could be processed with a 1mL monolith within 47min. The method compared to density gradient centrifugation has a 220-fold improvement in productivity.

The paper describes a method for the sensitive and selective determination of zearalenone and its metabolites in urine, plasma and faeces of horses by high performance liquid chromatography and atmospheric pressure chemical ionisation (APCI) mass spectrometry (MS). While only one step sample clean-up by an immunoaffinity column (IAC) was sufficient for plasma samples, urine and faeces samples had to be prepared by a combination of a solid-phase extraction (SPE) and an immunoaffinity column. The method allows the simultaneous determination of zearalenone and all of its metabolites; alpha-zearalenol, beta-zearalenol, alpha-zearalanol, beta-zearalanol and zearalanone. Dideuterated zearalanone was used as internal standard for quantification and the study of the matrix effect. Recovery rates between 56 and slightly above 100% were achieved in urine samples, and more than 80% in plasma and faeces samples. The limits of detection ranged from 0.1-0.5 microg/l or microg/kg, the limits of quantification from 0.5-1.0 microg/l or microg/kg. The practical use of the method is demonstrated by the analysis of spiked and naturally contaminated urine, plasma and faeces of horses.

The effect of pan-frying on the formation of cholesterol oxidation products (COPs) in different processed meat samples (beef patties, braised meat, and fillets of pork) was studied. Samples were pan-fried with or without addition of oil. Different unsaturated oils (olive oil, corn oil or partly hydrogenated plant oil) were used throughout the study. After extraction, seven toxicologically relevant COPs were analyzed using LC–MS. Prior to heat processing up to 6.7 mg COPs/kg extracted fat could be detected in the raw material. Neither the cholestanetriol nor 25-hydroxycholesterol, which are the most cytotoxic COPs in vitro, were detectable in any sample. Differences in the COPs contents were observed between beef (up to 16.5 mg/kg extracted fat) and pork (up to 22.2 mg/kg extracted fat) samples. In prepared samples higher COPs content was noted compared with raw samples. Generally, a certain order of COPs increase dependent on the plant oil used could be recognized: corn oil < partially hydrogenated plant oil < olive oil. It appears that short heating time, mild heating conditions, and the use of fresh and shortly stored raw materials keep COPs levels low.

A survey of zearalenone (ZON) in Indonesian maize-based food and feed was performed in 89 samples using an enzyme-linked immunosorbent assay (ELISA). Results indicated that 32 samples (36.0%) were contaminated in a range from 5.5 to 526 μg/kg. Only one highly contaminated sample was observed in a category of home-made food samples (>500 μg/kg) and the highest percentage of contaminated samples (>85.7%) was noted in a category of poultry feed. The positive samples based on the ELISA analyses were then confirmed using a high performance liquid chromatography (HPLC) method in combination with an immuno-affinity column for clean-up. Prior to sample measurement, the HPLC method was validated using a blank maize matrix. The overall average recovery of blank maize spiked with ZON at levels of 200–750 μg/kg were 100.2%. Detection limit based on a signal-to-noise ratio 3:1 was 3.0 μg/kg for ZON in maize samples and the linear range applicable for analyses was 5–900 μg/kg of ZON. Of 32 samples confirmed by HPLC, 25 samples were observed to be contaminated in a comparable range from 6.9 to 589 μg/kg. Comparative analysis of 25 positive samples using the ELISA and HPLC methods showed a reasonable correlation (R2=0.9258).

Haptoglobin (Hp) and immunoglobulins are plasma glycoproteins involved in the immune reaction of the organism after infection and/or inflammation. Porcine circovirus type 2-systemic disease (PCV2-SD), formerly known as postweaning multisystemic wasting syndrome (PMWS), is a globally spread pig disease of great economic impact. PCV2-SD affects the immunological system of pigs causing immunosuppression. The aim of this work was to characterize the Hp protein species of healthy and PCV2-SD affected pigs, as well as the protein backbone and the glycan chain composition of porcine Hp. PCV2-SD affected pigs had an increased overall Hp level, but it did not affect the ratio between Hp species. Glycoproteomic analysis of the Hp β subunits confirmed that porcine Hp is N-glycosylated and, unexpectedly, O-glycosylated, a PTM that is not found on Hp from healthy humans. The glyco-profile of porcine IgG and IgA heavy chains was also characterized; decreased levels of both proteins were found in the investigated group of PCV2-SD affected pigs. Obtained results indicate that no significant changes in the N- and O-glycosylation patterns of these major porcine plasma glycoproteins were detectable between healthy and PCV2-SD affected animals. Biological significance PCV2-SD is a disease of great economic importance for pig production, characterized by a complex response of the immune system. In the search of a better diagnostic/prognostic marker for porcine PCV2-SD, extensive analyses of the Hp protein backbone and the glycan chains were thoroughly analyzed by various techniques. This resulted in detection and confirmation of Hp O-glycosylation and the glyco-profiling of porcine IgG and IgA. The N- and O-glycosylation of these major porcine plasma glycoproteins appears to be not affected by PCV2-SD infection. Interestingly, these data suggest that this viral infection, which significantly affects the immune responses of the host, leaves the biosynthetic glycosylation processes in the liver and immune cells unaffected. Lack of PTM changes is in contrast to findings in humans where for both proteins pattern changes have been reported in several chronic and inflammatory diseases. This underlines the importance of studying species in detail and not reaching to conclusions by analogy. Furthermore, since Hp is usually quantified by immunoassays in clinical routine analyses, our findings indicate that no bias in Hp determination capabilities due to an altered carbohydrate pattern is to be expected.

Energy-rich diets can challenge metabolic and protective functions of the rumen epithelial cells, but the underlying factors are unclear. This study sought to evaluate proteomic changes of the rumen epithelium in goats fed a low, medium, or high energy diet. Expression of protein changes were compared by two-dimensional differential gel electrophoresis followed by protein identification with matrix assisted laser desorption ionisation tandem time-of-flight mass spectrometry. Of about 2,000 spots commonly detected in all gels, 64 spots were significantly regulated, which were traced back to 24 unique proteins. Interestingly, the expression profiles of several chaperone proteins with important cellular protective functions such as heat shock cognate 71 kDa protein, peroxiredoxin-6, serpin H1, protein disulfide-isomerase, and selenium-binding protein were collectively downregulated in response to high dietary energy supply. Similar regulation patterns were obtained for some other proteins involved in transport or metabolic functions. In contrast, metabolic enzymes like retinal dehydrogenase 1 and ATP synthase subunit beta, mitochondrial precursor were upregulated in response to high energy diet. Lower expressions of chaperone proteins in the rumen epithelial cells in response to high energy supply may suggest that these cells were less protected against the potentially harmful rumen toxic compounds, which might have consequences for rumen and systemic health. Our findings also suggest that energy-rich diets and the resulting acidotic insult may render rumen epithelial cells more vulnerable to cellular damage by attenuating their cell defense system, hence facilitating the impairment of rumen barrier function, typically observed in energy-rich fed ruminants.

Scope Patients with persistent egg allergy have more immunoglobulin E (IgE) against sequential than conformational epitopes of ovomucoid (OVO). Here, we aimed to identify compounds capable to render sequential epitopes in egg. Methods and results Glutathione was used for in vitro reduction of OVO and circular dichroism analyses were performed. Glutathione reduced OVO in a concentration-dependent manner. Egg white was analyzed for reduced proteins with a thiol probe and by MALDI-TOF/TOF. In unprocessed total egg white, several reduced proteins were detected by the thiol probe, among them reduced ovalbumin could be confirmed with MS analyses. Egg-allergics or sensitized controls were tested serologically (n = 19) for IgE against native and reduced OVO and in skin prick tests (n = 9). More patients had IgE against reduced than native OVO in Western blots. In skin prick test, five out of seven persistent egg-allergics and none of the controls reacted with reduced OVO. Conclusion Reduced egg proteins are present in natural egg white. Glutathione, which is present in egg and furthermore is used as texture-improving additive in processed food, is capable of reducing OVO. Patients with persistent egg allergy reacted rather to reduce the native OVO. Hence, our data indicate that reduction is a novel natural and processing-associated principle, which contributes to the allergenicity of food.

Automatic and manual sampling for ochratoxin A (OTA) in barley grain was compared under industrial conditions considering sampling uncertainty as well as practical and technical aspects. Ten tonnes of barley inoculated with Penicillium verrucosum were incubated until the OTA concentration reached approximately 15 µg kg(-1) and sampled with manual and automatic sampling. A nested experimental design and ANOVA was used to estimate variance components from sampling, sample reduction, sample preparation and analysis. Manual sampling resulted in a high sampling uncertainty and OTA concentrations in aggregate samples ranged from 2 to 80 µg kg(-1). When aggregate samples were formed by automatic sampling the uncertainty arising from nugget effects and spatial distribution was practically eliminated. Results from this study show that an automatic sampler mounted after a mixer or conveyer can provide representative samples of OTA from a moving stream of barley. Automatic sampling might present a practical and economical alternative to manual sampling for feed mill operators when monitoring low levels of mycotoxins in grain or other commodities. Despite careful precautions, sample preparation and analysis resulted in a relative uncertainty of ±40% (p = 0.95), which was attributed to the sub-sampling following the two grinding steps. Size fractionation of the coarsely ground barley showed that 40% of the total amount of OTA was present in a small fraction of fine particles with a strong tendency to aggregate or stick to equipment and containers. Thus, in order to take advantage of the automatic sampling, it is crucial to apply an appropriate sub-sampling to prevent segregation of particles which may affect the OTA measurements.

This paper describes the application of sol–gel immunoaffinity columns for clean up of ochratoxin A contaminated cereal crops. Monoclonal antibodies selective for OTA have been entrapped into the pores of a sol–gel matrix in order to prepare immunoaffinity columns. Different parameters such as amount of entrapped antibodies and loading conditions were optimized to obtain highest possible recoveries of OTA. The method has been found to be a suitable tool in sample preparation prior to HPLC-FLD determination and as selective as conventional commercially available immunoaffinity columns. In the clean up of different cereals mean recoveries of 82 ± 5%, 90 ± 6% and 91 ± 3%, were obtained for wheat, barley and rye, respectively, with sol–gel columns containing 1 mg of anti-OTA antibodies. The detection limit (signal-to-noise ratio, 3) was 0.5 μg/kg and the limit of quantification (signal-to-noise ratio, 10) determined to be 1 μg/kg. Sol–gel columns can be reused 7 times without significant loss of recovery. After 10 applications the recovery decreased to approx. 50%.

Risk assessment and monitoring of mycotoxins in agricultural commodities is associated with an increasing demand for appropriate analytical methods. Owing to the introduction of regulatory limits for various mycotoxins, the determination of mycotoxins in food and feed has become an important research issue in routine and governmental laboratories. The most prevalent methods for qualitative and quantitative determination, with the exception of enzyme linked immunosorbent assay (ELISA), are not able to analyse mycotoxins in complex matrices without sample pretreatment. In the same way as for all other analytes, sample preparation is one of the most crucial and important steps in the whole chain of analysis. There are different strategies used to determine mycotoxins in agricultural commodities as well as in biological samples, for example animal tissues, urine and so on. There are various approaches, such as solid phase extraction and liquid–liquid extraction; however, the most important and popular sample preparation technique used in mycotoxin analysis is immunoaffinity chromatography. In recent years there have been efforts to introduce selective and cost-effective strategies in sample pretreatment of mycotoxins. This chapter describes the significance of sample clean up in the analysis of major and abundant mycotoxins. Various conventional methods that are applied to sample preparation of mycotoxins in food, feed and biological samples will be discussed. Recent developments are included and different strategies for achieving more efficient extraction and separation are compared and presented.

The aim of the present study was to investigate the occurrence of mycotoxins in commercial dog food, as a basis to estimate the risk of adverse effects. Seventy-six dry dog food samples from 27 producers were purchased from retail shops, supermarkets, and specialized pet food shops in Vienna, Austria. The frequency and levels of deoxynivalenol (DON), zearalenone (ZEA), fumonisins (FUM), ochratoxin A (OTA). and aflatoxins (AF) in dry dog food were determined. Mycotoxin analysis were performed by commercial enzyme-linked immunosorbent assay (ELISA) test kits. Confirmatory analyses were done for DON, ZEA, and FUM by high performance liquid chromatography (HPLC) after extract clean-up with immunoaffinity columns. The correlations between ELISA and HPLC results for DON and ZEA were acceptable and indicated that ELISA could be a simple, low cost, and sensitive screening tool for mycotoxins detection, contributing to quality and safety of pet food. DON was the mycotoxin most frequently found (83% positives; median 308 µg/kg, maximum 1,390 µg/kg). ZEA (47% positives, median 51 µg/kg and maximum 298 µg/kg) and FUM (42% positives, median 122 µg/kg and maximum 568 µg/kg) were also frequently detected in dog food. OTA was less frequently found (5%, median 3.6 µg/kg, maximum 4.7 µg/kg. AF were not detected (0.5 µg/kg) in any sample. The results show that dry dog food marketed in Vienna are frequently contaminated with mycotoxins (DONZEAFUMOTA) in low concentrations, but do not contain AF. The high frequency of Fusarium toxins DON, ZEA, and FUM indicates the need for intensive control measures to prevent mycotoxins in dog foods. The mycotoxin levels found in dry dog food are considered as safe in aspects of acute mycotoxicoses. However, repeated and long-time exposure of dogs to low levels of mycotoxins may pose a health risk.

Plasmid DNA is being considered as a promising alternative to traditional protein vaccines or viral delivery methods for gene therapeutic applications. DNA-based products are highly flexible, stable, are easily stored and can be manufactured on a large scale. Although, much safer than viral approaches, issues have been raised with regard to safety due to possible integration of plasmid DNA into cellular DNA or spread of antibiotic resistance genes to intestinal bacteria by horizontal gene transfer. Accordingly, there is interest in methods for the production of plasmid DNA that lacks the antibiotic resistance gene to further improve their safety profile. Here, we report for the first time the gram-scale manufacturing of a minimized plasmid that is devoid of any additional sequence elements on the plasmid backbone, and merely consists of the target expression cassette and the bacterial origin of replication. Three different host/vector combinations were cultivated in a fed-batch fermentation process, comparing the progenitor strain JM108 to modified strains JM108murselect, hosting a plasmid either containing the aminoglycoside phosphotransferase which provides kanamycin resistance, or a marker-free variant of the same plasmid. The metabolic load exerted by expression of the aminoglycoside phosphotransferase was monitored by measuring ppGpp- and cAMP-levels. Moreover, we revealed that JM108 is deficient of the Lon protease and thereby refined the genotype of JM108. The main consequences of Lon-deficiency with regard to plasmid DNA production are discussed herein. Additionally, we found that the expression of the aminoglycoside phosphotransferase, conferring resistance to kanamycin, was very high in plasmid DNA producing processes that actually inclusion bodies were formed. Thereby, a severe metabolic load on the host cell was imposed, detrimental for overall plasmid yield. Hence, deleting the antibiotic resistance gene from the vector backbone is not only beneficial with regards to safety and potency of the end-product but also regarding the overall process performance.

The present paper describes the development of a new clean-up strategy for the analysis of aflatoxins (AFs) in food. The sample preparation method is based on immuno-ultrafiltration (IUF) which, in contrast to immunoaffinity chromatography, makes use of antibodies in free form. After selecting an appropriate ultrafiltration (UF) device and optimizing different operation conditions the IUF method was applied to the clean-up of maize and rice. Quantification of AFs was carried out by HPLC and fluorescence detection, after postcolumn derivatization in a Kobracell. The IUF method was shown to be as selective as sample clean-up using commercial immunoaffinity columns. Recovery rates and RSD for the AFs G(2), G(1), B(2) and B(1) in spiked rice were found to be 76 +/- 3, 76 +/- 2, 83 +/- 5 and 99 +/- 14%, respectively. The analysis of a FAPAS (food analysis performance assessment scheme) maize material resulted in AFs concentrations which were in the range assigned by the producer of the reference material.

The paper describes a sample clean-up method for the co-isolation of deoxynivalenol (DON) and zearalenone (ZON), two mycotoxins naturally co-occurring in wheat. The method is based on immunoaffinity columns prepared by co-immobilising anti-DON and anti-ZON antibodies in a porous sol–gel glass. The main task in developing the method consisted in finding a loading medium allowing retention of both analytes as well as a common elution medium for the dissociation of both antigen–antibody complexes formed. This can be achieved by co-extracting DON and ZON with ACN–water (60:40, v/v), reducing the acetonitril concentration to 2.5% before loading an aliquot of the diluted sample extract onto the DON/ZON column. The columns are washed with 5 ml of MeOH–water (10:90, v/v) before DON and ZON are co-eluted with 4 ml of ACN–water (50:50, v/v). Concentrations of DON and ZON are determined with HPLC-UV and HPLC-fluorescence detection, respectively. The sample clean-up method was shown to be applicable to wheat and wheat products, e.g., cornflakes, milk wheat mash and rusk. Spiking experiments (spike level 500 μg DON/kg and 50 μg ZON/kg) resulted in recovery rates from 82% to 111%.

Mucorales are regarded as the aetiological agents of Mucormycosis. Their capabilities to produce mycotoxins are not profoundly investigated, in contrast to those of the fungi from the generaPenicillium, Aspergillus, orFusarium. The aim of this study was to isolate and identify fungi of the order Mucorales and investigate mycotoxins production. Twelve samples of visibly moulded grass silage and eight samples of damaged whole crop maize silage were analysed. Malt extract agar plates were used for sub cultivation. Three fungal species of the order Mucorales were isolated from grass silage, which were identified by their macro-and micro-morphology asAbsidia corymbifera, Mucor circinelloides andRhizopus stolonifer. The cytotoxicity ofMucor circinelloides extract was analysed using the cytotoxicity test (MTT assay) and the result, showed a low cytotoxicity. Additionally extracts fromAbsidia corymbifera, Mucor circinelloides andRhizopus stolonifer were tested for mycotoxin-production using an LC/MS/MS-based multimycotoxin method. 3-nitropropionic acid was detected in the culture extract ofMucor circinelloides.

An experiment was carried out to examine the effects of feeding Fusarium toxin-contaminated wheat (8.21 mg deoxynivalenol (DON) and 0.09 mg zearalenone (ZON) per kg dry matter) at different feed intake levels on the biotransformation and carry-over of DON in dairy cows. For this purpose, 14 ruminal and duodenal fistulated dairy cows were fed a diet containing 60% concentrate with a wheat portion of 55% (Fusarium toxin-contaminated wheat (mycotoxin period) or control wheat (control period)) and the ration was completed with maize- and grass silage (50 : 50) on a dry matter basis. Daily DON intakes ranged from 16.6 to 75.6 mg in the mycotoxin period at dry matter intakes of 5.6-20.5 kg. DON was almost completely biotransformed to de-epoxy DON (94-99%) independent of the DON/feed intake, and the flow of DON and de-epoxy DON at the duodenum related to DON intake ranged from 12 to 77% when the Fusarium toxin-contaminated wheat was fed. In the serum samples, de-epoxy DON was detected in the range of 4-28 ng ml-1 in the mycotoxin period, while concentrations of DON were all below the detection limit. The daily excretion of DON and de-epoxy DON in the milk of cows fed the contaminated wheat varied between 1 and 10 microg and between 14 and 104 microg, respectively. The total carry-over rates as the ratio between the daily excretion of DON and de-epoxy DON into milk and DON intake were in the ranges of 0.0001-0.0002 and 0.0004-0.0024, respectively. Total carry-over rates of DON as DON and de-epoxy DON into the milk increased significantly with increasing milk yield. In the urine samples, de-epoxy DON was the predominant substance as compared with DON with a portion of the total DON plus de-epoxy DON concentration to 96% when the Fusarium toxin-contaminated wheat was fed, whereas the total residues of DON plus de-epoxy DON in faeces ranged between 2 and 18% of DON intake in the mycotoxin period. The degree of glucuronidation of de-epoxy DON was found to be approximately 100% in serum. From 33 to 80% of DON and from 73 to 92% of de-epoxy DON, and from 21 to 92% of DON and from 86 to 100% of de-epoxy DON were glucuronidated in the milk and urine, respectively. It is concluded that DON is very rapidly biotransformed to de-epoxy DON in the rumen and only negligible amounts of DON and de-epoxy DON were transmitted into the milk within the range of 5.6-20.5 kg day-1 dry matter intake and milk yields (fat corrected milk) between 10 and 42 kg day-1.

The aim of the present study was to examine the effects of feeding Fusarium toxin-contaminated wheat to dairy cows on nutrient utilization in the rumen and on duodenal flow of deoxynivalenol (DON), zearalenone (ZON) and their metabolites. Six dairy cows fitted with a large rumen cannula and a simple T-shaped cannula at the proximal duodenum was used in two experiments. The experiments included a control period in which the uncontaminated control wheat was fed and a period in which the control wheat was replaced by the Fusarium toxin-contaminated wheat (8.05 and 7.15 mg DON/kg and 0.26 and 0.1 mg ZON/kg in Expts 1 and 2 respectively). The wheat portion of the daily ration amounted to 50% on a dry matter (DM) basis and rations were completed with hay or grass silage. Five of the six cows were non-lactating and the total daily DM-intake ranged between 4 and 12 kg. The pH-values and the concentration of volatile fatty acids in ruminal fluid were not significantly influenced by feeding the contaminated wheat. In contrast, the postprandial ammonia concentration was consistently higher when the mycotoxin-contaminated wheat was fed. Moreover, the flow of microbial protein and utilizable protein at the duodenum were reduced at the same time. The concentrations of DON and ZON and of their metabolites in freeze-dried duodenal digesta were either not detectable or negligible during the control periods whereas distinct concentrations were measured during the periods where the contaminated wheat was fed. DON was nearly completely metabolized to de-epoxy-DON and the flow at the duodenum ranged between 4% and 28% of DON-intake. The ZON metabolites alpha-zearalenol (ZOL) and beta-ZOL were recovered at the duodenum beside the parent toxin ZON. Their recovery as a percentage of ZON-intake ranged between 43% and 132%. In conclusion, feeding of Fusarium toxin-contaminated wheat altered the ruminal protein utilization. The question of whether this effect was a result of the mycotoxin being present in the rumen or of Fusarium growth-related structural (cell wall) changes of the wheat grain needs to be clarified. The low recovery of DON at the duodenum would indicate either a nearly complete degradation of the molecule in the rumen or an absorption by the mucosa of the rumen, whereas the higher ZON recovery would suggest a lower degradation of the parent toxin in the rumen and/or recovery of some bile-originating entero-hepatic cycling ZON/metabolites.

Twenty-eight herbal medicinal products from Thailand were investigated for aflatoxin (AF) contaminations by employing a specific HPLC assay for the determination of AFB1, B2, G1 and G2. The samples were extracted with 80% (v/v) methanol in water before further cleaned up with an immunoaffinity column and followed by the detection of AFs by using an electrochemically post-column derivatization with iodine and fluorescence detector. The extraction procedure was optimized in order to obtain the best recovery. The method was successfully carried out with all the herbal products diversified as to compositions and dosage forms. The results revealed that five (18%) of herbal samples were contaminated with detectable amount of the total AFs ranging from 1.7 to 14.3 ng/g. The association between particular herbal/plant and the AF contaminated could not be determined due to the low frequency of positive samples. The contaminated products were those in tablet (4) and capsule (1) dosage forms. It was possible that the original fungal infection of these products may have been derived from either the crude herbal or other ingredients making these preparations, such as starch. In conclusion, none of the AF contaminated level found was above the current legislative level permissible in Thailand (20 ng/g). A word of caution, however, exporting some high AF-contaminated herbal products to countries where more stringent permissable level of aflatoxins exist could result in trade Barriers.

Salmon fillets were steamed, or pan-fried without oil, with olive oil, with corn oil, or with partially hydrogenated plant oil. The exchange between the salmon and the pan-frying oils was marginal, but it was detectable as slight modifications in the fatty acid pattern and the tocopherol contents according to the oil used. Primary and secondary oxidation products were only slightly increased or remained unchanged, which indicated a slight lipid oxidation effect due to the heating procedures applied. The same was observed for tocopherol levels, which remained almost stable and were not affected by the oxidation process. The sum of cholesterol oxidation products (COPs) increased after the heating processes from 0.9 microg/g in the raw sample to 6.0, 4.0, 4.4, 3.3, and 9.9 microg/g extracted fat in pan-fried without oil, with olive oil, corn oil, partially hydrogenated plant oil, and steamed, respectively. A highly significant correlation was found between the fatty acid pattern and the total amount of COPs (r2 = 0.973, p < 0.001). No change has been determined in the n-3 fatty acids content and in the polyunsaturated/saturated-ratio of the cooked salmon fillets. Moderate pan-frying (6 min total) and steaming (12 min) of salmon did not accelerate lipid oxidation but significantly increased the content of COPs. The highest increase of COPs was found through steaming, mainly due to the longer heat exposure. The used frying oils did not influence the outcome; no significant difference between heat treatment with or without oil has been determined.

A selective analytical method based on high-performance liquid chromatography (HPLC), combined with atmospheric pressure chemical ionisation (APCI-) mass spectrometry (MS), has been developed for simultaneous determination of B-trichothecenes and the major metabolites of deoxynivalenol. The method allows simultaneous analysis of nivalenol (NIV), deoxynivalenol (DON), 15-acetyldeoxynivalenol (15-AcDON), 3-acetyldeoxynivalenol (3-AcDON), fusarenon X (Fus-X) and de-epoxydeoxynivalenol (DOM-1). The method is based on one-step sample clean-up using a multifunctional MycoSep column. A linear gradient mobile phase system, consisting of water:acetonitrile:methanol (H2O:ACN:MeOH) at a flow-rate of 1 ml/min, and a Polar-RP C18 column, were utilised to obtain the best resolution of all tested compounds along with column and equilibrating within 30 min. Dexamethasone (Dex) was used as internal standard. The developed method shows good repeatability for inter- and intra-day precisions as well as good linearity of calibration curves (r2 ranged from 0.9936 to 0.9998). Average recoveries for tested compounds in both matrices have been determined ranging from 63.7 to 102.3% and limit of quantification (LOQ) ranged from 25 to 150 ng/g. The utility and practical impact of the method is demonstrated using contaminated pig urine and maize samples.

Deoxynivalenol (DON) is a common mycotoxin contaminant in feedstuffs. It has been shown to cause diverse toxic effects in animals. The aim of the present study was to evaluate the effects of DON on the glucose transport capacity in chickens' jejunum and to investigate the permeation of DON itself by the Ussing chamber technique. Glucose uptake into chicken jejunal epithelia was measured after the addition of 200 mumol/L of (14)C-labeled glucose to the mucosal solution. Glucose uptake under control condition was 3.28 +/- 0.53 nmol/cm(2) x min. The contribution of sodium glucose-linked transporter 1 (SGLT-1) to total glucose uptake was estimated by inhibiting SGLT-1 with phlorizin (100 micromol/L). In the presence of phlorizin, glucose uptake was reduced (P < 0.05) to 1.21 +/- 0.19 nmol/cm(2) x min. Deoxynivalenol decreased (P < 0.05) the glucose uptake in the absence of phlorizin to 1.81 +/- 0.24 nmol/cm(2) x min but had no additional effect on the glucose uptake in the presence of phlorizin (0.97 +/- 0.17 nmol/cm(2) x min). Mucosal-to-serosal permeation of DON was proportional to the initial DON concentration over a concentration range from 1 to 10 mug/mL on the mucosal side. Apparent permeability at 10 microg/mL of DON measured 60 to 90 min after DON application was 1.7 x 10(-05) cm/s. It can be concluded that DON (10 mg/L) decreases glucose uptake almost as efficiently as phlorizin. The similarity between the effects of phlorizin and DON on glucose uptake evidences their common ability to inhibit Na(+)-D-glucose cotransport. In addition to local effects, DON can be absorbed from the jejunum. A predominant part of DON passes across the chicken intestinal epithelium by passive diffusion, which is likely on the paracellular pathway. The results imply that the exposure to DON-contaminated feeds may negatively affect animal health and performance by local (i.e., inhibition of intestinal SGLT-1) and systemic effects.