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ABSTRACT:A monoclonal-based ELISA, coupled with an assay buffer, solvent and solid phase extraction procedures, was validated for use in the monitoring of egg samples for 3-amino-2-oxazolidinone (AOZ). The procedures allow the detection of protein bound AOZ in the form of 2-nitrophenyl derivative (NPAOZ) in sample supernatant or extract after acid hydrolysis and derivatisation with o-nitrobenzaldehyde. The assays were validated according to criteria set down by Commission Decision (2003) for the performance and validation of analytical methods for chemical residues. The detection capability of ELISA's for AOZ in eggs (set on the basis of acceptance of no false negatives) was 0.6, 0.3 and 0.3 µg/kg for buffer, solvent and solid phase extraction, respectively. These values are well below the maximum required performance limit (MRLP) of 1 µg/kg for tissue bound residues of nitrofuran antibiotics. An excellent correlation of results (r = 0.99, n = 14) obtained by the ELISA and LC-MS/MS techniques within the concentration range of 0-5 µg/kg was found in the incurred egg samples. The eggs collected from layer chickens fed 30 and 400 mg/kg of furazolidone for 10 days were monitored by ELISA until AOZ concentrations approached the LoD.

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Deoxynivalenol (DON)-contaminated feed represents a serious problem for pigs due to their high sensitivity to its toxicological effects. The aim of the present study was to evaluate the impact of intrauterine DON exposure on the immune system of piglets. Pure DON was intravenously administered to sows at the end of gestation (during the last 2–3 days of gestation, one dose of 300 µg per day). The plasma concentration of DON was analyzed using liquid chromatography combined with high-resolution Orbitrap-based mass spectrometry (LC–MS/MS (HR)) and selected immune parameters were monitored six times in piglets from birth to 18 weeks. DON was found in the plasma of 90% of newborn piglets at a mean concentration of 6.28 ng/mL and subsequently, at one, three, and seven weeks after birth with decreasing concentrations. Trace amounts were still present in the plasma 14 weeks after birth. Flow cytometry revealed a significant impact of DON on T lymphocyte subpopulations during the early postnatal period. Lower percentages of regulatory T cells, T helper lymphocytes, and their double positive CD4+CD8+ subset were followed by increased percentages of cytotoxic T lymphocytes and γδ T cells. The capacity to produce pro-inflammatory cytokines was also significantly lower after intrauterine DON exposure. In conclusion, this study revealed a long-term persistence of DON in the plasma of the piglets as a consequence of short-term intrauterine exposure, leading to altered immune parameters.

A sensitive and selective analytical method for the determination of four thyreostats (tapazol, thiouracil, methylthiouracil and propylthiouracil) in cow's milk, lamb's milk, and goat's milk was developed and validated according to 2002/657/EC criteria. Proteins in milk samples were precipitated by acetonitrile and analytes were derivatised with 3-iodobenzylbromide. Afterwards, derivatives were separated from the matrix by liquid-liquid extraction with ethyl acetate as an organic solvent and analysis was carried out using LC-MS/MS in a positive electrospray mode. The method provides, for all determined analytes, decision limits CCα below 1 ng ml(-1) and a detection capability CCβ value below 1.5 ng ml(-1). The stability of analytes in sample extracts stored at various conditions was also tested and evaluated.

Deoxynivalenol (DON) is a mycotoxin frequently found in cereals, and pigs are one of the most sensitive farm species to DON. The aim of this study was to determine the effects of DON in very low doses on peripheral blood mononuclear cells (PBMC) and on particular lymphocyte subpopulations. The cells were exposed to 1, 10 and 100 ng/mL of DON and lymphocyte viability, proliferation, and cytokine (Interleukin (IL)-1β, IL-2, IL-8, IL-17, Interferon (IFN) γ and tumor necrosis factor (TNF) α production were studied. Cells exposed to DON for 5 days in concentrations of 1 and 10 ng/mL showed higher viability compared to control cells. After 18 h of DON (100 ng/mL) exposure, a significantly lower proliferation after mitogen stimulation was observed. In contrast, an increase of spontaneous proliferation induced by DON (100 ng/mL) was detected. After DON exposure, the expression of cytokine genes decreased, with the exception of IL-1β and IL-8, which increased after 18 h exposure to 100 ng/mL of DON. Among lymphocyte subpopulations, helper T-cells and γδ T-cells exhibiting lower production of IL-17, IFNγ and TNFα were most affected by DON exposure (10 ng/mL). These findings show that subclinical doses of DON lead to changes in immune response.

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This study aimed to obtain a comprehensive look at the influence of castration on mRNA expression of the genes CYP2E1, CYP1A2, CYP2A19, HSD3B, SULT2A1 and SULT1A1 and their correlation with boar taint compounds (androstenone, skatole and indole) and Improvac-specific antibodies in a Czech commercial hybrid (Large White × Landrace (sow) × Duroc (boar)). Pigs were divided into groups of entire male pigs (NC), pigs castrated surgically (SC), pigs immunologically castrated and slaughtered 8 weeks (IM8) or 15 weeks (IM15) after the second dose of Improvac, and gilts (GI). Hepatic mRNA expression, measured by quantitative real-time polymerase chain reaction, differed significantly between the control group (entire male pigs) and all groups of interest for CYP2E1, CYP1A2 and CYP2A19. The mRNA level of the HSD3B gene differed significantly between the control group and the IM8, IM15 and GI groups. SULT1A1 gene expression was significantly different between the control group and the SC, IM8 and GI. In the case of SULT2A1, a significant difference was observed only between the control group and IM8 pigs. For all genes and treatment groups described above, expression was increased relative to the control. Significant differences for Improvac-specific antibodies between IM8 and IM15 groups were observed, indicating decrease of antibodies over time. Moreover, negative correlations between androstenone and mRNA levels of CYP2A19, CYP2E1 and SULT1A1 suggest that gene expression is suppressed.