Siegrid De Baere

Toxicokinetic study and absolute oral bioavailability of deoxynivalenol, T-2 toxin and zearalenone in broiler chickens

Mycotoxins lead to economic losses in animal production. A way to counteract mycotoxicosis is the use of detoxifiers. The European Food Safety Authority stated that the efficacy of detoxifiers should be investigated based on toxicokinetic studies. Little information is available on the absolute oral bioavailability and the toxicokinetic parameters of deoxynivalenol, T-2 and zearalenone in broilers. Toxins were administered intravenously and orally in a two-way cross-over design. For deoxynivalenol a bolus of 0.75mg/kg BW was administered, for T-2 toxin 0.02mg/kg BW and for zearalenone 0.3mg/kg BW. Blood was collected at several time points. Plasma levels of the mycotoxins and their metabolite(s) were quantified using LC-MS/MS methods and toxicokinetic parameters were analyzed. Deoxynivalenol has a low absolute oral bioavailability (19.3%). For zearalenone and T-2 no plasma levels above the limit of quantification were observed after an oral bolus. Volumes of distribution were recorded, i.e. 4.99, 0.14 and 22.26L/kg for deoxynivalenol, T-2 toxin and zearalenone, respectively. Total body clearance was 0.12, 0.03 and 0.48L/minkg for deoxynivalenol, T-2 toxin and zearalenone, respectively. After IV administration, T-2 toxin had the shortest elimination half-life (3.9min), followed by deoxynivalenol (27.9min) and zearalenone (31.8min).

Inhibition tests for detection and presumptive identification of tetracyclines, beta-lactam antibiotics and quinolones in poultry meat.

A combination of three plates, seeded with strains of Micrococcus luteus, Bacillus cereus or Escherichia coli, can be used for detection of residues of betalactam antibiotics, tetracyclines and fluoroquinolones. The sensitivity of each plate is optimal for only one of these groups, resulting in detection limits (LOD) lower than the corresponding maximum residue limits (MRL) and in distinct inhibition patterns typical for each antibiotic family. Beta-lactam antibiotics such as penicillin G, ampicillin and amoxicillin give only inhibition zones on the plate with M. luteus. Tetracyclines are detected up to the MRL level with B. cereus, and fluoroquinolones with E. coli. The LODs of the antibiotics tested were as follows: penicillin G (PENG) 0.9 ng, ampicillin (AMPI) 0.6 ng and amoxicillin (AMOX) 1.0 ng on the plate with M. luteus; tetracycline (TET) 4 ng, oxytetracycline (OXY) 3 ng, doxycycline (DOX) 0.6 ng, and chlortetracycline (CHL) 0.3 ng on the plate with B. cereus; enrofloxacin (ENRX) 1.5 ng, ciprofloxacin (CIPX) 0.5 ng and flumequine (FLUM) 1.5 ng on the plate with E. coli. The combination of plates enables the laboratory to select appropriate chromatographic techniques for identification and quantification of the residues. On the other hand, the three groups can also be detected on one plate seeded with Bacillus subtilis, although the limits of detection are higher: PENG 0.4 ng, AMPI and AMOX 3 ng; TET 5 ng, OXY 8 ng, DOX 1 ng, CHL 0.5 ng, ENRX 4 ng, CIPX 10 ng and FLUM 4 ng. The test was applied to 228 broiler fillets and to 27 turkey thighs, originating from different poultry slaughterhouses. Nineteen broiler fillets contained inhibiting substances. The positive results of the inhibition tests were confirmed with a chromatographic technique. Doxycycline residues were found in 16 samples and amoxicillin in two.

Quantitative determination of several toxicological important mycotoxins in pig plasma using multi-mycotoxin and analyte-specific high performance liquid chromatography-tandem mass spectrometric methods.

A sensitive and reliable multi-mycotoxin method was developed for the identification and quantification of several toxicological important mycotoxins such as deoxynivalenol (DON), deepoxy-deoxynivalenol (DOM-1), T-2 toxin (T-2), HT-2 toxin (HT-2), zearalenone (ZON), zearalanone (ZAN), α-zearalenol (α-ZOL), β-zearalenol (β-ZOL), α-zearalanol (α-ZAL), β-zearalanol (β-ZAL), ochratoxin A (OTA), fumonisin B1 (FB1) and aflatoxin B1 (AFB1) in pig plasma using liquid chromatography combined with heated electrospray ionization triple quadrupole tandem mass spectrometry (LC-h-ESI-MS/MS). Sample clean-up consisted of a deproteinization step using acetonitrile, followed by evaporation of the supernatant and resuspension of the dry residue in water/methanol (85/15, v/v). Each plasma sample was analyzed twice, i.e. once in the ESI+ and ESI- mode, respectively. This method can be used for the assessment of animal exposure to mycotoxins and in the diagnosis of mycotoxicoses. For the performance of toxicokinetic studies with individual mycotoxins, highly sensitive analyte-specific LC-MS/MS methods were developed. The multi-mycotoxin and analyte-specific methods were in-house validated: matrix-matched calibration graphs were prepared for all compounds and correlation and goodness-of-fit coefficients ranged between 0.9974-0.9999 and 2.4-15.5%, respectively. The within- and between-run precision and accuracy were evaluated and the results fell within the ranges specified. The limits of quantification for the multi-mycotoxin and analyte-specific methods ranged from 2 to 10 ng/mL and 0.5 to 5 ng/mL, respectively, whereas limits of detection fell between 0.01-0.52 ng/mL and <0.01-0.15 ng/mL, respectively.

Modified Fusarium mycotoxins unmasked: from occurrence in cereals to animal and human excretion

Modified mycotoxins formed by plants, fungi and during some food processing steps may remain undetected by analytical methods, potentially causing underestimation of mycotoxin exposure and risk. Furthermore, due to altered physico-chemical characteristics of modified mycotoxins, these compounds might have different gastro-intestinal absorption compared to the unmodified forms, leading to altered modified mycotoxin plasma concentrations. Additionally, modified mycotoxins can be converted back into their corresponding unmodified forms by in vivo hydrolysis upon oral ingestion. This review aims to describe the current knowledge on the production, occurrence, toxicity and toxicokinetic properties of the modified Fusarium mycotoxins. The need for more occurrence data to correctly assess the risks associated with these modified mycotoxins is clearly indicated, including differences between commodities as well as geographical and climatological influences. Research on toxicity of these modified forms demonstrates the possibility of significant decreases as well as increases in the toxic effects of these compounds compared with those of the unmodified forms. Their toxicokinetics demonstrates that a decreased (increased) polarity of modified mycotoxins might cause enhanced (decreased) oral absorption. The possibility of in vivo hydrolysis, altered toxicity and their wide-spread occurrence makes modified mycotoxins a complex threat for which a risk assessment will require prospective multi-disciplinary efforts.

Butyrate production in phylogenetically diverse Firmicutes isolated from the chicken caecum

Sixteen butyrate‐producing bacteria were isolated from the caecal content of chickens and analysed phylogenetically. They did not represent a coherent phylogenetic group, but were allied to four different lineages in the Firmicutes phylum. Fourteen strains appeared to represent novel species, based on a level of ≤ 98.5% 16S rRNA gene sequence similarity towards their nearest validly named neighbours. The highest butyrate concentrations were produced by the strains belonging to clostridial clusters IV and XIVa, clusters which are predominant in the chicken caecal microbiota. In only one of the 16 strains tested, the butyrate kinase operon could be amplified, while the butyryl‐CoA : acetate CoA‐transferase gene was detected in eight strains belonging to clostridial clusters IV, XIVa and XIVb. None of the clostridial cluster XVI isolates carried this gene based on degenerate PCR analyses. However, another CoA‐transferase gene more similar to propionate CoA‐transferase was detected in the majority of the clostridial cluster XVI isolates. Since this gene is located directly downstream of the remaining butyrate pathway genes in several human cluster XVI bacteria, it may be involved in butyrate formation in these bacteria. The present study indicates that butyrate producers related to cluster XVI may play a more important role in the chicken gut than in the human gut.

Determination of gentamicin in swine and calf tissues by high-performance liquid chromatography combined with electrospray ionization mass spectrometry.

Gentamicin is a broad-spectrum aminoglycoside antibiotic widely used in veterinary medicine for the treatment of serious infections. The purpose of this study was to develop and validate a method to determine gentamicin residues in edible tissues of swine and calf. Extraction of gentamicin was performed using a liquid extraction with phosphate buffer containing trichloroacetic acid, followed by a solid-phase clean-up procedure on a CBA weak cation-exchange column. Tobramycin was used as the internal standard. After drying of the eluate, the residue was redissolved and further analyzed by reversed-phase liquid chromatography/electrospray ionization tandem mass spectrometry (MS/MS). Chromatographic separation of the internal standard tobramycin and the gentamicin components was achieved on a Nucleosil (5 microm) column using a mixture of 10 mM pentafluoropropionic acid in water and acetonitrile as the mobile phase. The gentamicin components C1a, C2 + C2a and C1 could be identified with the MS/MS detection, and subsequently quantified. The method was validated according to the requirements of the EC at the maximum residue limit (MRL) (100 ng g(-1) for muscle and fat, 200 ng g(-1) for liver and 1000 ng g(-1) for kidney), half the MRL and double the MRL levels. Calibration graphs were prepared for all tissues and good linearity was achieved over the concentration ranges tested (r > 0.99 and goodness of fit <10%). Limits of quantification of 25.0 ng g(-1) were obtained for the determination of gentamicin in muscle, fat, liver and kidney tissues of swine and calf, which correspond in all cases to at least half the MRLs. Limits of detection ranged between 0.5 and 2.5 ng g(-1) for the tissues. The within-day and between-day precisions (RSD) and the results for accuracy fell within the ranges specified. The method was successfully used for the determination of gentamicin in tissue samples of swines and calves medicated with gentamicin by intramuscular injection.

Quantitative analysis of oxytetracycline and its 4-epimer in calf tissues by high-performance liquid chromatography combined with positive electrospray ionization mass spectrometry

Tetracycline antibiotics are commonly used in veterinary medicine because of their broad spectrum activity and cost effectiveness. Oxytetracycline (OTC) is one of the most important members of this antibiotic family. The purpose of this study was to develop and validate a method to determine OTC residues in edible tissues of calf. Extraction of OTC and its 4-epimer (4-epiOTC), in the presence of the internal standard demethylchlortetracycline (DMCTC), was performed using a liquid extraction with sodium succinate solution (pH 4.0), followed by protein removal with trichloroacetic acid and paper filtration. Further solid-phase extraction clean-up on an HLB polymeric reversed phase column was performed to obtain an extract suitable for LC-MS-MS analysis. Chromatographic separation of the internal standard, and especially OTC and its 4-epimer, was achieved on a PLRP-S polymeric reversed phase column, using a mixture of 0.001 M of oxalic acid, 0.5% (v/v) of formic acid and 3% (v/v) of tetrahydrofuran in water (mobile phase A) and tetrahydrofuran (mobile phase B) as the mobile phase, and at a column temperature of 60 degrees C. OTC and its 4-epimer could be identified using the MS-MS detection technique, and were subsequently quantified. The method has been validated according to the requirements of the EC at the MRL (maximum residue limit, 100 ng g(-1) for muscle, 300 ng g(-1) for liver and600 ng g(-1) for kidney), half the MRL and double the MRL levels, as well for OTC as for 4-epiOTC. Calibration graphs were prepared for all tissues and good linearity was achieved over the concentration ranges tested (r > 0.99 and goodness of fit < 10%). Limits of quantification of half the MRLs were obtained for the analysis of OTC and 4-epiOTC in muscle, liver and kidney tissues of calf. Limits of detection ranged for both components between 0.8 and 48.2 ng g(-1). The within-day and between-day precisions, expressed as RSD values, were all below the maximum allowed RSD values calculated according to the Horwitz equation. The results for accuracy fell within the -20% to +10% range. Recoveries were between 47 and 56% for OTC, and between 52 and 62% for 4-epiOTC, depending on the tissue. The method has been successfully used for the quantitative determination of OTC and 4-epiOTC in tissue samples of calves medicated with OTC by intramuscular injection.

Porcine intestinal epithelial barrier disruption by the Fusarium mycotoxins deoxynivalenol and T-2 toxin promotes transepithelial passage of doxycycline and paromomycin

BackgroundThe gastrointestinal tract is the first target for the potentially harmful effects of mycotoxins after intake of mycotoxin contaminated food or feed. With deoxynivalenol (DON), T-2 toxin (T-2), fumonisin B1 (FB1) and zearalenone (ZEA) being important Fusarium toxins in the northern hemisphere, this study aimed to investigate in vitro the toxic effect of these mycotoxins on intestinal porcine epithelial cells derived from the jejunum (IPEC-J2 cells). Viability of IPEC-J2 cells as well as the proportion of apoptotic and necrotic IPEC-J2 cells was determined by flow cytometry after 72 h of exposure to the toxins. Correlatively, the integrity of the intestinal epithelial cell monolayer was studied using Transwell® inserts, in which the trans-epithelial electrical resistance (TEER) and passage of the antibiotics doxycycline and paromomycin were used as endpoints.ResultsWe demonstrated that the percentage of Annexin-V-FITC and PI negative (viable) cells, Annexin-V-FITC positive and PI negative (apoptotic) cells and Annexin-V-FITC and PI positive (necrotic) IPEC-J2 cells showed a mycotoxin concentration-dependent relationship with T-2 toxin being the most toxic. Moreover, the ratio between Annexin-V-FITC positive and PI negative cells and Annexin-V-FITC and PI positive cells varied depending on the type of toxin. More Annexin-V-FITC and PI positive cells could be found after treatment with T-2 toxin, while more Annexin-V-FITC positive and PI negative cells were found after exposure to DON. Consistent with the cytotoxicity results, both DON and T-2 decreased TEER and increased cellular permeability to doxycycline and paromomycin in a time- and concentration-dependent manner.ConclusionsIt was concluded that Fusarium mycotoxins may severely disturb the intestinal epithelial barrier and promote passage of antibiotics.

Quantitative determination of the Fusarium mycotoxins beauvericin, enniatin A, A1, B and B1 in pig plasma using high performance liquid chromatography–tandem mass spectrometry

A sensitive and reliable method was developed for the identification and quantification of beauvericin, enniatin A, A1, B and B1 in pig plasma using liquid chromatography combined with heated electrospray ionization tandem mass spectrometry. Sample clean-up consisted of a deproteinization step using acetonitrile, followed by evaporation of the supernatant and resuspension of the dry residue in acetonitrile/water (80/20, v/v). The method was in-house validated: matrix-matched calibration graphs were prepared for all compounds and correlation and goodness-of-fit coefficients ranged between 0.9980 and 0.9995 and between 5.2% and 11.3%, respectively. The within- and between-run precision and accuracy were evaluated and the results fell within the ranges specified. The limits of quantification were 0.1 ng/mL for enniatin A and A1 and 0.2 ng/mL for beauvericin, enniatin B and B1, whereas limits of detection were ≤ 10 pg/mL for all analytes. The method has been applied for the analysis of real plasma samples from one pig that received an oral bolus (0.05 mg/kg BW) of the investigated mycotoxins. At the applied dosage, the results indicated the suitability of the method for use in toxicokinetic studies with enniatins.

Practical approach for the stability testing of veterinary drugs in solutions and in biological matrices during storage

According to the SANCO/1805/2000 document and the EMEA/CVMP/573/00 guideline, one of the performance characteristics to be determined for an analytical method is the analyte stability. A practical approach is presented for the stability testing of veterinary drugs in solvents and in biological matrices during storage. These drugs comprise analytes belonging to the classes of antibiotics, sulfonamide chemotherapeutics, anthelminthics and non-steroidal anti-inflammatory drugs. Examples of the stability in solution are given for clindamycin, ketoprofen, oxytetracycline, 4-epioxytetracycline, sulfadiazine, trimethoprim, tylosin A and their respective internal standards. For the stability in matrix during storage, plasma or tissues are fortified with the analyte. Results of the recovery after storage are shown for amoxycillin in pig tissue, doxycyclin, sulfachloropyridazin and trimethoprim in broiler tissue and clindamycin, closantel, doxycyclin and ketoprofen in animal plasma. The analysis of incurred plasma samples containing closantel revealed that the temperature at which the samples are processed is critical. Compounds are analyzed by liquid chromatography with UV or tandem mass spectrometric detection.