Natural contamination of Manitoba barley by 3,15-diacetyldeoxynivalenol and its detection by immunochromatography.

Contamination of Canadian barley samples by 3,15-diacetyldeoxynivalenol was detected by enzyme immunoassays combined with liquid chromatography-mass spectrometry. This is the first reported natural occurrence of this mycotoxin. The barley was infected mainly with Fusarium graminearum. Deoxynivalenol, 3-acetyldeoxynivalenol, and zearalenone were also found.     

First survey on the natural occurrence of Fusarium mycotoxins in Bulgarian wheat

Wheat for human consumption (140 samples) was collected after harvest from all regions of Bulgaria. The 1995 crop year was characterized by heavy rainfall in the spring and summer months. The internal mycoflora of wheat samples was dominated by Fusarium spp. and Alternaria spp., and storage fungi were rarely present. The samples were analysed for contamination with Fusarium mycotoxins deoxynivalenol (DON), 3-acetyldeoxynivalenol (3-AcDON), 15-acetyldeoxynivalenol (15-AcDON), T-2 Toxin (T-2), diacetoxyscirpenol (DAS), and zearalenone (ZEA), using enzyme immunoassay methods. DON and ZEA were the predominant toxins, with a contamination frequency of 67% and 69%, respectively. The average levels of these toxins in positive samples were 180 g/kg (DON) and 17 g/kg (ZEA), maximum concentrations were 1800 g kg^–1 and 120 g kg^–1, respectively. Acetyl derivatives of DON, namely 3-AcDON and 15-AcDON, were found in 2.1 % and 0.7% of the samples, at at maximum level of about 100 g kg^–1. Only one sample was positive for T-2 (55 g/kg), DAS was not detected. This is the first report about the natural occurrence of a range of Fusarium mycotoxins in wheat for human consumption in Bulgaria.Abbreviations 3-AcDON 3-acetyldeoxynivalenol - 15-AcDON 15-acetyldeoxynivalenol - DAS diacetoxyscirpenol - DON deoxynivalenol - EIA enzyme immunoassay - T-2 T-2 toxin - ZEA zearalenone     

Trichothecenes and Mycoflora in Wheat Harvested in Nine Locations in Buenos Aires Province, Argentina

A total of 120 freshly harvested wheat samples from the 2004 season in nine locations from Northern Buenos Aires Province, Argentina, were analysed for trichothecene natural occurrence and associated mycoflora, and for determining the influence of commonly used fungicide field treatment and the cultivar type on trichothecene contamination. The trichothecenes T-2 tetraol, T-2 triol, HT-2 and T-2 toxin (HT-2, T-2), diacetoxyscirpenol (DAS), nivalenol (NIV), deoxynivalenol (DON), 3-acetyldeoxynivalenol (3-ADON) and 15-acetyldeoxynivalenol (15-ADON) were analysed by gas chromatography and electron capture detection. Detection limits ranged from 4 to 20 μg/kg. The isolation frequencies of species were calculated. Alternaria alternata, Fusarium graminearum, Fusarium poae and Fusarium semitectum were the predominant fungal species identified as endogenous mycoflora. The type of cultivar and the fungicide field treatment did not affect significantly the trichothecene contamination. The trichothecenes type A detected were HT-2 and T-2 triol toxins and the type B were DON, NIV and 3-ADON. Based on 120 samples the incidences were 21.7% for 3-ADON, 22.5% for HT-2, 27.5% for T-2 triol and 85% for DON. NIV was confirmed in one sample. Mean levels of trichothecene positive samples were between 7 and 2788 μg/kg.     

Deoxynivalenol and 3-acetyldeoxynivalenol — mycotoxins associated with wheat head fusariosis in Poland

Samples of wheat naturally infected in the field byFusarium culmorum (W.G.Sm.) Sacc. andFusarium graminearum Schwabe were analyzed for deoxynivalenol, 3-acetyldeoxynivalenol, and zearalenone. No zearalenone was detected at levels higher than 0.5mg/kg. Deoxynivalenol was present in 100% and 3-acetyldeoxynivalenol in 80% of the examined samples at levels of 0.21 to 30.4 mg/kg and 0.54 to 29.54 mg/kg, respectively. The mycotoxin levels in the chaff were 5 to 50 times higher than in the kernels. This is the first report on natural occurrence of 3-acetyldeoxynivalenol in wheat. This toxin, in addition to deoxynivalenol, was highly correlated with wheat head fusariosis. These findings suggest that more attention should be given to the occurrence of 3-acetyldeoxynivalenol in cereal grains during the growth as well as during storage.     

Europium Nanoparticle-Based High Performing Immunoassay for the Screening of Treponemal Antibodies

Treponema pallidum subspecies pallidum (Tp) is the causative agent of syphilis which mainly spreads through sexual contact, blood transfusion and perinatal route. In order to curtail the spread of the infection and to clinically manage the disease, timely, accurate and reliable diagnosis is very important. We have developed an immunoassay for the detection of treponemal antibodies in human serum or plasma samples. In vivo biotinylated and non-biotinylated versions of the recombinant antigen were designed by the fusion of three Tp-specific antigens namely Tp15, Tp17 and Tp47. These fusion antigens were expressed in E. coli and purified using single-step metal affinity chromatography. Biotinylated fusion antigen immobilized on streptavidin coated plate was used to capture the treponemal antibodies and the non-biotinylated antigen coated on europium nanoparticles was used as tracer. Assays with two different incubation times of 10 min and 1 h were developed, and following the incubation the europium fluorescence was measured using time-resolved fluorometry. The developed time-resolved fluorometric (TRF) immunoassays were evaluated with in-house and commercial serum/plasma sample panels. For well-established treponemal antibodies positive or negative samples, the sensitivity of TRF immunoassay with 10 min incubation time was 97.4%, and of TRF immunoassay with 1 h incubation time was 98.7%, and the specificities of both the TRF immunoassays were 99.2%. For the samples with discordant results with the reference assays, both the TRF immunoassays showed better specificity than the Enzygnost syphilis enzyme immunoassay as a screening test. The two different incubation times did not have any significant effect on the signal to cutoff (S/Co) ratios obtained with the two immunoassays (p = 0.06). Our results indicate that the developed immunoassay with a short incubation time of 10 min has the potential to be used in clinical laboratories and in blood-bank settings as a screening test for treponemal antibodies.     

Therapeutic efficacy of intra-articular adrenomedullin injection in antigen-induced arthritis in rabbits

Introduction

Autoantibodies to the ribosomal P proteins represent a highly specific marker for the diagnosis of systemic lupus erythematosus, where they have been associated with certain clinical manifestations. Historically, autoantibodies against ribosomal P proteins have been detected by indirect immunofluorescence, immunodiffusion, immunoblot, and other immunoassays. More recently, enzyme-linked immunosorbent assays and line and addressable laser bead immunoassays have become more widely used. The primary goal of this study was to determine the sensitivity of indirect immunofluorescence using conventional HEp-2 substrates in the detection of sera with ribosomal P antibodies as detected by other immunoassays.

Methods

Anti-ribosomal P-positive sera (n = 345) as detected by an addressable laser bead immunoassay were collected between 2003 and 2007 and analysed by indirect immunofluorescence. Furthermore, 51 anti-ribosomal P-positive samples from an unselected systemic lupus erythematosus cohort (n = 100) and the Centers for Disease Control and Prevention (CDC) anti-nuclear antibody (ANA) reference sera were tested for anti-ribosomal P reactivity.

Results

In the cohort of 345 anti-ribosomal P-positive samples identified by addressable laser bead immunoassay, a low sensitivity (<30%) of indirect immunofluorescence on HEp-2 cell substrates was observed. Although the degree of sensitivity varied among different manufacturers, all immunofluorescence substrates exhibited limited sensitivity and false-negative results were not restricted to samples with low anti-ribosomal P titers. Even the anti-ribosomal P reactivity of CDC ANA reference serum number 12 was not clearly predictable by indirect immunofluorescence. Comparison of five different methods for the detection of anti-ribosomal P found moderate qualitative agreements.

Conclusions

Based on our data, we conclude that indirect immunofluorescence on HEp-2 cells is not a reliable screening test for the prediction of ribosomal P antibodies. As this method is widely used as a first-line screening test for anti-nuclear and other autoantibodies, special considerations for the detection of ribosomal P antibodies are needed. As with many other autoantibodies, further effort is required for the standardisation of ribosomal P immunoassays.     

Natural occurrence of deoxynivalenol, 3-acetyl-deoxynivalenol, 15-acetyl-deoxynivalenol,nivalenol, 4,7-dideoxynivalenol,and zearalenone in polish wheat

Three wheat samples collected in 1987 in Central Poland and naturally infected withFusarium spp were analyzed for the presence ofFusarium spp andFusarium toxins. Heads were separated into three fractions: kernels with visibleFusarium damage, healthy looking kernels, and chaff + rachis. The samples contained deoxynivalenol (2.0 – 40.0μg/g), nivalenol (O.O1μg/g), 4,7-dideoxynivalenol (0.10 – 0.15μg/g). 15-acetyldeoxynivalenol (0.10–2.00 μg/g), 3-acetyldeoxynivalenol (O/1Oμg/g), and zearalenone (0.01–2.00μg/g). This is the first report about 15 - acetyldeoxynivalenol in European wheat and the co-occurrence of 3 - acetyldeoxynivalenol and 15-acetyldeoxynivalenol in the same sample of contaminated cereals.     

Microwave oven and boiling waterbath extraction of hepatotoxins from cyanobacterial cells

Low-cost, straightforward methods for the extraction of microcystins and nodularins from cyanobacterial cells were developed using a microwave oven and boiling waterbath. The use of organic solvents, such as methanol, which can interfere with sensitive analytical procedures, e.g. immunoassays, can thus be avoided. Analysis by protein phosphatase inhibition assay and high performance liquid chromatography indicated that purified microcystin-LR was unaffected by the microwave oven and boiling waterbath treatments. Four microcystins of differing hydrophobicities were successfully extracted from Microcystis PCC 7813 by both treatments at yields equivalent to those obtained by longer protocols using methanol. Assessment of the microwave oven and boiling waterbath extraction methods with laboratory strains and environmental samples of cyanobacteria showed good correlation with results from lyophilisation and methanol extraction, when extracts were analysed by high performance liquid chromatography with diode array detection (R(2)>/=0.92). The microwave and boiling waterbath extraction methods also sterilised the environmental bloom samples, as evidenced by the abolition of heterotrophic bacterial growth.     

Immunoenzyme demonstration of viruses and virus-specific antibodies by using conjugates based on gene engineering-produced beta-lactamase

Enzyme immunoassays for the detection of viral antigens and virus-specific antibodies in biological samples have been described. Molecular complexes of antibodies and beta-lactamase (penicillinase) have been used as anti-specific conjugates. To synthesize the conjugate, the enzyme obtained with the aid of genetic engineering has been used. Enzyme immunoassays have been tested for the indication of the influenza virus and virus-induced specific antibodies. Enzyme immunoassays were shown to possess certain advantages (e.g., the use of simple and nontoxic substrate) along with the sensitivity identical to that of other methods, employing peroxidase-based conjugates.     

The occurrence of culmorin and hydroxy-culmorins in cereals

Forty-five samples from 1988–1995 of naturally contaminated grain, barley, wheat and oats, three samples of mixed feed, and 16 samples of grain artificially inoculated with Fusarium culmorum during the flowering stage were analysed for deoxynivalenol (DON), 3-acetyldeoxynivalenol (3-acetyl-DON), culmorin and hydroxy-culmorins. These compounds are secondary metabolites produced by the fungal species F. culmorum and F. graminearum. Acetonitrile-water extract of the samples was purified on a Mycosep^TM#225 column, derivetized using pentafluoropropionic anhydride (PFPA) and analysed by gas chromatography-mass spectrometry (GC-MS). The amount of each of culmorin, 5-, 12-, 14 and 15-hydroxy-culmorin and one unknown hydroxy-culmorin were determined relative to the amount of DON plus 3-acetyl DON for each sample. The ratio between the total amount of culmorin compounds and the DON compounds ranged from 0.14 to 1.07 in the samples. This study shows that there is a strong correlation between the amount of DON present in the grain and the amount of culmorin and hydroxy-culmorins present. The ratio of each of the culmorin compounds relative to the amount of DON compounds were in the same range in the grain artificially inoculated by F. culmorum as found in an earlier study for F. culmorum strains cultivated on rice, while the hydroxy-culmorin profile in the naturally contaminated grain was more similar to what was found for the F. graminearum cultures in the same study [1]. These results indicate that F. graminearum may be a relatively important source for DON in grain also in relatively cold areas. This revised version was published online in June 2006 with corrections to the Cover Date.     

Natural occurrence of Fusarium toxins in barley harvested during five years in an area of southwest Germany

A total of 44, 40, 47, 51, and 58 barley samples for feed use were collected randomly after the 1987, 1989, 1990, 1991, and 1992 crops, respectively, from farms located in an area of southwest Germany. The sum of precipitation from May to September was high in 1987 and markedly lower in 1989–1992. Deoxynivalenol, 3-. and 15-acetyldeoxynivalenol, nivalenol, fusarenon-X, T-2 toxin, HT-2 toxin and diacetxyscirpenol were determined by gas chromatography with mass selective detection (GC-MS), zearalenone,α- and β-zearalenol by GC-MS or by HPLC. Deoxynivalenol was the major toxin with incidences at 71–98% and mean contents at 42–400 μg/kg. In contrast, incidences of zearalenone, 3-acetyldeoxynivalenol, nivalenol, HT-2 toxin, and T-2 toxin were at 7–68, 7–48, 11–41, 0–9, and 2–29%, respectively; with mean contents at 3–146 μg/kg. α- and β-zearalenol and diacetoxyscirpenol were not detected in any sample. 15-acetyldeoxynivalenol and fusarenon-X were assayed in samples from 1987, 1991 and 1992. 15-acetyldeoxynivalenol was detected in 30, 0 and 2% of samples, respectively, with an average content of positive samples at 8 and 4 μg/kg, fusarenon-X was not detected. Over the years, incidences and levels of toxins remained constant, decreased or increased. The correlation between the occurrence of toxins and level of precipitation is discussed. This revised version was published online in June 2006 with corrections to the Cover Date.     

Natural Occurrence of 16 Fusarium Toxins in Grains and Feedstuffs of Plant Origin from Germany

A total of 220 samples comprising cereals, cereal byproducts, corn plants and corn silage as well as non-grain based feedstuffs was randomly collected during 2000 and 2001 from sources located in Germany and analysed for 16 Fusarium toxins. The trichothecenes scirpentriol (SCIRP), 15-monoacetoxyscirpenol (MAS), diacetoxyscirpenol (DAS), T-2 tetraol, T-2 triol, HT-2 and T-2 toxin (HT-2, T-2), neosolaniol (NEO), deoxynivalenol (DON), 3-acetyldeoxynivalenol (3-ADON), 15-acetyldeoxynivealenol (15-ADON), nivalenol (NIV) and fusarenon-X (FUS-X) were determined by gas chromatography/mass spectrometry. Zearalenone (ZEA) and α- and β-zearalenol (α- and β-ZOL) were analysed by high performance liquid chromatography with fluorescence and UV-detection. Detection limits ranged between 1 and 19 μg/kg. Out of 125 samples of a group consisting of wheat, oats, corn, corn byproducts, corn plants and corn silage only two wheat samples did not contain any of the toxins analysed. Based on 125 samples the incidences were at 2–11% for DAS, NEO, T-2 Triol, FUS-X, α- and β-ZOL, at 20–22% for SCIRP, MAS, T-2 tetraol and 3-ADON, at 44–74% for HT-2, T-2, 15-ADON, NIV and ZEA, and at 94% for DON. Mean levels of positive samples were between 6 and 758 μg/kg. Out of 95 samples of a group consisting of hay, lupines, peas, soya meal, rapeseed meal and other oilseed meals, 64 samples were toxin negative. DAS, T-2 triol, NEO and FUS-X were not detected in any sample. The incidences of DON and ZEA were at 14 and 23% respectively, those of the other toxins between 1–4%, mean levels of positive samples were between 5 and 95 μg/kg.     

Rapid screening method for determination of Ecstasy and amphetamines in urine samples using gas chromatography-chemical ionisation mass spectrometry

The need for analytical screening tests more reliable and valid to detect amphetamine and related "designer drugs" in biological samples is becoming critical, due to the increasing diffusion of these drugs on the European illegal market. The most common screening procedures based on immunoassays suffer a number of limitations, including low sensitivity, lack of specificity and limited number of detectable substances. This paper describes a screening method based on gas-chromatography-mass-spectrometry (GC/MS) using positive chemical ionisation (PCI) detection. Methanol was used as reactant gas in the ionisation chamber. Molecular ions of different compounds were monitored, allowing a sensitivity of 5-10 ng/ml with high selectivity. The sensitivity of the method gives positive results in samples taken 48-72 h after intake of one dose of 50-100 mg. The method is simple and rapid. Sample preparation was limited to one liquid-liquid extraction, without any hydrolysis and derivatisation. Hydrolysis is critical to identify metabolites excreted as conjugates. Blank urine samples spiked with known amounts of amphetamine (AM), methylamphetamine (MA), methylenedioxyamphetamine (MDA), methylenedioxymethylamphetamine (MDMA), methylenedioxyethylamphetamine (MDEA) and methylenedioxyphenyl-N-methyl-2-butanamine (MBDB) were analysed. The method was successfully tested on real samples of urine from people, whose use of amphetamine was suspected, and results were compared with results obtained with immunoassays.     

Elimination of botulinum neurotoxin (BoNT) type B from drinking water by small-scale (personal-use) water purification devices and detection of BoNT in water samples

Seven small-scale drinking water purification devices were evaluated for their capacity to eliminate botulinum neurotoxin (BoNT) type B from drinking water. Influent water inoculated with toxic Clostridium botulinum cultures and effluent purified water samples were tested for the presence of BoNT by using a standard mouse bioassay and two commercial rapid enzyme immunoassays (EIAs). The water purification devices based on filtration through ceramic or membrane filters with a pore size of 0.2 to 0.4 microm or irradiation from a low-pressure UV-lamp (254 nm) failed to remove BoNT from raw water (reduction of < 0.1 log10 units). A single device based on reverse osmosis was capable of removing the BoNT to a level below the detection limit of the mouse bioassay (reduction of > 2.3 log10 units). The rapid EIAs intended for the detection of BoNT from various types of samples failed to detect BoNT from aqueous samples containing an estimated concentration of BoNT of 396,000 ng/liter.     

玉米培养物中弯角镰孢菌代谢产物的分析与鉴定

从致牛烂蹄病的稻草中分离的弯角镰孢菌菌株(Fusarium camptoceras)于1988年和1991年分两批接种玉米中,培养物用乙腈:水(3:1,v/v)提取,正已烷脱脂,Florisil色谱柱净化,经净化的提取液经电子捕获气相色谱(GC-ECD)、薄层色谱(TLC)分析和气相色谱-质谱联用(GC-MS)鉴定,证实了弯角镰孢菌可同时产生多种单端孢霉烯族化合物。弯角镰孢菌能同时产生这些代谢物,在国内外未见报道。     

Susceptibility of Barley Cultivars and Lines to Fusarium Infection and Mycotoxin Accumulation in Kernels

Heads of 12 barley genotypes (8 cultivars and 4 lines) were inoculated with conidial suspension of the following single isolates: F. culmorum no. 3, F. graminearum no. 122 and F. sporotrichioides no. ATCC 62 360. The number of kernels per head. 1000 Kernel weight and yield have been calculated for each genotype. Seed samples collected at harvest were analysed for each genotype. Seed samples collected at harvest were analysed for several trichothecene mycotoxins and zearalenone.The mycotoxin concentrations (mg/kg) in barley kernels inoculated with F. graminearum were as follows. deoxynivalenol (DON) 0.1 to 5.4 (av. 2.3). 3-acetyldeoxy-nivalenol (3-AcDON) 0.0–0.2 (av. 0.1), 15-acetyldeoxynivalenol (15-AcDON) 0.0–0.7 (av.0.2), nivalenol (NIV) 0.0–0.8 (av. 0.3). zearalenone (ZEA) 0.0–0.1 (av. 0.0); F. culmorum: DON 0.6 to 12.0 (av. 5.3), 3-AcDON 0.1 to 1.0 (av. 0.6). 15-AcDON nd. NIV 0.1–0.7 (av. 0.3). ZEA 0.1–0.5 (av. 0.2). F. sporotrichioides T-2 toxin 2.4–13.9 (av. 6.0), HT-2-toxin 0.1–0.8 (av.0.3) and neosolaniol 0.2–1.5 (av.0.7).     

Simultaneous determination of major B-trichothecenes and the de-epoxy-metabolite of deoxynivalenol in pig urine and maize using high-performance liquid chromatography-mass spectrometry

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.     

The use of a CCD imaging luminometer in the quantitation of luminogenic immunoassays

We describe the application of a CCD Imaging Luminometer (CCDIL) for the detection and quantitation of rapid, simultaneous, peroxidase-linked chemiluminescent immunoassays of multiple samples of human serum alphafetoprotein (AFP). Results from some analogous immunoassays (total IgE, and thyroid stimulating hormone (TSH)) are included for comparison. Values for precision and antigen concentration obtained using the CCDIL and colorimetric versions of the immunoassays, on human serum samples, were in good agreement. The flexibility of the CCDIL is demonstrated; its ability to detect and quantitate antigen (particularly AFP) on a variety of solid phases is indicated. The work on the AFP immunoassays illustrates not only the flexibility of the CCDIL for sample presentation on a variety of solid phase systems, but also some relative merits of such systems.     

Solid phase enzyme immunoassays for the quantification of serum amyloid P (SAP) and complement component 3 (C3) proteins in acute-phase mouse sera.

Simple, reliable and sensitive enzyme immunoassays have been developed for the quantification of the mouse acute-phase SAP and C3 proteins. The ELISA systems were validated using sera from mice injected with S. dysenteriae endotoxin, and detected 500 pg protein/ml. The assays use 96-well microtitre plates permitting rapid processing of a large number of samples.     

Elimination of Botulinum Neurotoxin (BoNT) Type B from Drinking Water by Small-Scale (Personal-Use) Water Purification Devices and Detection of BoNT in Water Samples

Seven small-scale drinking water purification devices were evaluated for their capacity to eliminate botulinum neurotoxin (BoNT) type B from drinking water. Influent water inoculated with toxic Clostridium botulinum cultures and effluent purified water samples were tested for the presence of BoNT by using a standard mouse bioassay and two commercial rapid enzyme immunoassays (EIAs). The water purification devices based on filtration through ceramic or membrane filters with a pore size of 0.2 to 0.4 μm or irradiation from a low-pressure UV-lamp (254 nm) failed to remove BoNT from raw water (reduction of <0.1 log₁₀ units). A single device based on reverse osmosis was capable of removing the BoNT to a level below the detection limit of the mouse bioassay (reduction of >2.3 log₁₀ units). The rapid EIAs intended for the detection of BoNT from various types of samples failed to detect BoNT from aqueous samples containing an estimated concentration of BoNT of 396,000 ng/liter.