Multi-residue analysis of 20 mycotoxins including major metabolites and emerging mycotoxins in freshwater using UHPLC-MS/MS and application to freshwater ponds in flanders, Belgium.

Mycotoxins are known for their negative impact on human and animal health as they frequently contaminate food and feed products from crop origin that are consumed by humans and animals. Furthermore, mycotoxins can leach out of plant tissue, to be transported through runoff water into nearby ponds where they can exert negative effects on aquatic organisms, such as fish, amphibians and zooplankton. The overall goal of this study was to develop a SPE-UHPLC-MS/MS method for the detection and quantification of multiple mycotoxins in amphibian breeding ponds. The method was validated and yielded acceptable within-run and between-run apparent recoveries and precision, as well as good linearity. Matrix effects (i.e. 75.7-109.6%, ≤ 17.8% RSD) were evaluated using water from 20 different ponds in Flanders, Belgium. By incorporating internal standards, overall results improved and adequate precision values (i.e. ≤ 15%) were obtained according to the EMA guideline. Additionally, extraction recovery (n = 3) was evaluated, yielding good results for all mycotoxins (i.e. 75.3-109.1%, ≤15% RSD), except for AME (i.e. 6.7 ± 0.7%), which implied the need for a matrix-matched calibration curve. Detection sensitivity was in the low nanograms per liter range. Storage stability experiments indicated that sample storage at 4 °C in amber glass bottles and analysis performed within 96 h after sampling was sufficient to avoid loss by degradation for all compounds, excluding ß-ZAL and ß-ZEL, for which analysis within 24 h is more indicated. The method was successfully applied to water samples originating from 18 amphibian breeding ponds situated across Flanders. Overall, enniatins B, B1 and A1 were most commonly detected at maximum concentrations of 6.9, 3.3 and 2.6 ng L-1, respectively, followed by detection of beauvericin (1.1 ng L-1 and < 1 ng L-1), alternariol monomethyl ether (< 10 ng L-1), HT2-toxin (< 40 ng L-1), zearalenone (< 25 ng L-1) and α-zearalanol (< 10 ng L-1). We believe that this method will boost further research into the dynamics and ecotoxicological impact of mycotoxins in aquatic environments.

Selection and transfer of an IncI1-tet(A) plasmid of Escherichia coli in an ex vivo model of the porcine caecum at doxycycline concentrations caused by crosscontaminated feed.

AIMS: The aim of this study was to investigate the effect of subtherapeutic intestinal doxycycline (DOX) concentrations (4 and 1 mg l-1 ), caused by cross-contamination of feed, on the enrichment of a DOX-resistant commensal Escherichia coli and its resistance plasmid in an ex vivo model of the porcine caecum. METHODS AND RESULTS: A DOX-resistant, tet(A)-carrying, porcine commensal E. coli strain (EC 682) was cultivated for 6 days in the porcine caecum model under different conditions (0, 1 and 4 mg l-1 DOX). EC 682, other coliforms and anaerobic bacteria were enumerated daily. A selection of isolated DOX-resistant coliforms (n = 454) was characterized by rep-PCR clustering, PCR assays (Inc1 and tet(A)) and micro broth dilution susceptibility tests (Sensititre). Both 1 and 4 mg l-1 DOX-enriched medium had a significantly higher selective effect on EC 682 and other resistant coliforms than medium without DOX. Transconjugants of EC 682 were isolated more frequently in the presence of 1 and 4 mg l-1 DOX compared to medium without DOX. CONCLUSIONS: Subtherapeutic intestinal DOX concentrations have the potential to select for DOX-resistant E. coli, and promote the selection of transconjugants in a porcine caecum model. SIGNIFICANCE AND IMPACT OF THE STUDY: Cross-contamination of feed with antimicrobials such as DOX likely promotes the spread of antimicrobial resistance. Therefore, it is important to develop or fine-tune guidelines for the safe use of antimicrobials in animal feed and its storage.

Effects of Xylo-Oligosaccharides on Broiler Chicken Performance and Microbiota.

In broiler chickens, feed additives, including prebiotics, are widely used to improve gut health and to stimulate performance. Xylo-oligosaccharides (XOS) are hydrolytic degradation products of arabinoxylans that can be fermented by the gut microbiota. In the current study, we aimed to analyze the prebiotic properties of XOS when added to the broiler diet. Administration of XOS to chickens, in addition to a wheat-rye-based diet, significantly improved the feed conversion ratio. XOS significantly increased villus length in the ileum. It also significantly increased numbers of lactobacilli in the colon and Clostridium cluster XIVa in the ceca. Moreover, the number of gene copies encoding the key bacterial enzyme for butyrate production, butyryl-coenzyme A (butyryl-CoA):acetate CoA transferase, was significantly increased in the ceca of chickens administered XOS. In this group of chickens, at the species level, Lactobacillus crispatus and Anaerostipes butyraticus were significantly increased in abundance in the colon and cecum, respectively. In vitro fermentation of XOS revealed cross-feeding between L. crispatus and A. butyraticus. Lactate, produced by L. crispatus during XOS fermentation, was utilized by the butyrate-producing Anaerostipes species. These data show the beneficial effects of XOS on broiler performance when added to the feed, which potentially can be explained by stimulation of butyrate-producing bacteria through cross-feeding of lactate and subsequent effects of butyrate on gastrointestinal function.

Enantioselective pharmacokinetics of ketoprofen in calves after intramuscular administration of a racemic mixture.

The pharmacokinetic properties of ketoprofen were determined in 4-week-old calves after intramuscular (i.m.) injection of a racemic mixture at a dose of 3 mg/kg body weight. Due to possible enantioselective disposition kinetics and chiral inversion, the plasma concentrations of the R(-) and S(+) enantiomer were quantified separately, using a stereospecific HPLC-UV assay. A distinct predominance of the S(+) enantiomer was observed, as well as significantly different pharmacokinetic parameters between R(-) and S(+) ketoprofen. More in specific, a greater value for the mean area under the plasma concentration-time curve (AUC(0→∞)) (46.92 ± 7.75 and 11.13 ± 2.18 µg·h/mL for the S(+) and R(-) enantiomer, respectively), a lower apparent clearance (Cl/F) (32.8 ± 5.7 and 139.0 ± 25.1 mL/h·kg for the S(+) and R(-) enantiomer, respectively) and a lower apparent volume of distribution (V(d)/F) (139 ± 14.7 and 496 ± 139.4 mL/kg for the S(+) and R(-) enantiomer, respectively) were calculated for the S(+) enantiomer, indicating enantioselective pharmacokinetics for ketoprofen in calves following i.m. administration.

Pharmacokinetics of oral transmucosal and intramuscular dexmedetomidine combined with buprenorphine in cats.

Plasma concentrations and pharmacokinetics of dexmedetomidine and buprenorphine after oral transmucosal (OTM) and intramuscular (i.m.) administration of their combination in healthy adult cats were compared. According to a crossover protocol (1-month washout), a combination of dexmedetomidine (40 µg/kg) and buprenorphine (20 µg/kg) was given OTM (buccal cavity) or i.m. (quadriceps muscle) in six female neutered cats. Plasma samples were collected through a jugular catheter during a 24-h period. Plasma dexmedetomidine and buprenorphine concentrations were determined by liquid chromatography-tandem mass spectrometry. Plasma concentration-time data were fitted to compartmental models. For dexmedetomidine and buprenorphine, the area under the plasma concentration-time curve (AUC) and the maximum plasma concentrations (Cmax ) were significantly lower following OTM than following i.m. administration. For buprenorphine, time to reach Cmax was also significantly longer after OTM administration than after i.m. injection. Data suggested that dexmedetomidine (40 µg/kg) combined with buprenorphine (20 µg/kg) is not as well absorbed from the buccal mucosa site as from the intramuscular injection site.

Pharmacokinetics of gamithromycin after intravenous and subcutaneous administration in broiler chickens.

Gamithromycin is a new macrolide antibiotic that is only registered for use in cattle to treat respiratory disorders such as bovine respiratory disease. The aim of this study was to determine the pharmacokinetics of gamithromycin in broiler chickens. Gamithromycin (6 mg/kg of BW) was injected intravenously (IV) or subcutaneously (SC) to six 4-wk-old chickens in a parallel study design, and blood was collected at different time points postadministration. Quantification of gamithromycin in plasma was performed using an in-house validated liquid chromatography-tandem mass spectrometry method and the pharmacokinetics analyzed according to a 2-compartmental model. Following IV administration, the mean area under the plasma concentration-time curve (AUC0→∞), and α and ß half-life of elimination (t1/2el α and t1/2el ß) were 3,998 h•ng/mL, 0.90 h, and 14.12 h, respectively. Similar values were obtained after a SC bolus injection, i.e., 4,095 h•ng/mL, 0.34 h, and 11.63 h, for AUC0→∞, t1/2el α, and t1/2el ß, respectively. The mean maximum plasma concentration (889.46 ng/mL) appeared at 0.13 h. Gamithromycin showed a large volume of distribution after IV as well as SC administration, 27.08 and 20.89 L/kg, respectively, and a total body clearance of 1.61 and 1.77 L/h•kg, respectively. The absolute bioavailability was 102.4%, showing that there is a complete absorption of gamithromycin after a SC bolus injection of 6 mg/kg of BW.

Hepatic and intestinal CYP3A expression and activity in broilers.

Cytochrome P450 is involved in drug metabolism. Subfamily CYP3A shows a degree of similarity across different animal species. However, little information is available about its expression and activity in broiler chickens. A RT-PCR method was developed for the quantification of CYP3A37 expression in the liver and small intestine of broilers. A higher expression in the jejunum was observed compared with that in the ileum. In the liver, a significantly lower expression compared with that in the jejunum was noticed. Thus, the role of the small bowel in drug metabolism cannot be neglected in broilers. CYP3A activity was studied in vitro using midazolam as a substrate. Two protocols for the preparation of intestinal microsomes were compared. Mincing of the tissues before ultracentrifugation seemed to be more appropriate than a protocol based on ethylenediaminetetra-acetic acid separation. CYP3A activity revealed to be the highest in the duodenum with a decreasing trend towards the ileum. Activity in liver was comparable to duodenal activity.

Chronic exposure to the mycotoxin T-2 promotes oral absorption of chlortetracycline in pigs.

The aim of this study was to investigate whether T-2 toxin, a potent Fusarium mycotoxin, affects the oral absorption of the antibiotic chlortetracycline in pigs. Animals were allocated to blank feed without T-2 toxin (controls), feed containing 111 µg T-2/kg feed, T-2-contaminated feed supplemented with a yeast-derived feed additive, or blank feed supplemented solely with the feed additive, respectively. After 21 days, an intragastric bolus of chlortetracycline was given to assess potential alterations in the pharmacokinetics of this commonly used antibiotic. A significantly higher area under the plasma concentration-time curve and maximal plasma concentration of chlortetracycline was observed after intake of T-2-contaminated feed compared with control. Thus, exposure to T-2-contaminated feed can influence the oral bioavailability of chlortetracycline. This effect could have consequences for the withdrawal time of the drug and the occurrence of undesirable residues in edible tissues.

Transfer of flubendazole and tylosin from feed at cross-contamination levels to various poultry matrices.

Residues of veterinary drugs and feed additives used extensively in animal husbandry are sometimes found in edible matrices. In this study, broilers received experimental feed, containing either flubendazole or tylosin, at cross-contamination levels of 2.5%, 5%, and 10% of the therapeutic dose to determine the transfer ratio of these molecules from feed to poultry matrices. Breast and thigh muscle and liver samples were collected during treatment and depletion periods and then analyzed using liquid chromatography-tandem mass spectrometry. The parent molecule flubendazole and its 2 major metabolites were quantified. After 3 to 5 d, a plateau phase was reached, and a few days after withdrawal of the experimental feed, a depletion of residues was noted. Significant difference between both muscle types was noted for flubendazole. Strong metabolization of flubendazole in the liver was seen. For tylosin, no residue concentrations above the limit of quantification could be detected in muscle. None of the residue concentrations for either molecule exceeded the corresponding maximum residue limits.

Transfer of flubendazole and tylosin at cross contamination levels in the feed to egg matrices and distribution between egg yolk and egg white.

Chemical residues may be present in eggs from laying hens' exposure to drugs or contaminants. These residues may pose risks to human health. In this study, laying hens received experimental feed containing flubendazole or tylosin at cross contamination levels of 2.5, 5, and 10% of the therapeutic dose. Eggs were collected daily and analysis of the whole egg, egg white, and egg yolk was performed using liquid chromatography tandem mass spectrometry. Highest concentrations of the parent molecule flubendazole, as well as the hydrolyzed and the reduced metabolite, were detected in egg yolk. Residue concentrations of the parent molecule were higher compared with those of the metabolites in all egg matrices. No tylosin residue concentrations were detected above the limit of quantification for all concentration groups and in all egg matrices. Neither molecule exceeded the set maximum residue limits.

Efficacy and safety testing of mycotoxin-detoxifying agents in broilers following the European Food Safety Authority guidelines.

Contamination of feeds with mycotoxins is a worldwide problem and mycotoxin-detoxifying agents are used to decrease their negative effect. The European Food Safety Authority recently stated guidelines and end-points for the efficacy testing of detoxifiers. Our study revealed that plasma concentrations of deoxynivalenol and deepoxy-deoxynivalenol were too low to assess efficacy of 2 commercially available mycotoxin-detoxifying agents against deoxynivalenol after 3 wk of continuous feeding of this mycotoxin at concentrations of 2.44±0.70 mg/kg of feed and 7.54±2.20 mg/kg of feed in broilers. This correlates with the poor absorption of deoxynivalenol in poultry. A safety study with 2 commercially available detoxifying agents and veterinary drugs showed innovative results with regard to the pharmacokinetics of 2 antibiotics after oral dosing in the drinking water. The plasma and kidney tissue concentrations of oxytetracycline were significantly higher in broilers receiving a biotransforming agent in the feed compared with control birds. For amoxicillin, the plasma concentrations were significantly higher for broilers receiving an adsorbing agent in comparison to birds receiving the biotransforming agent, but not to the control group. Mycotoxin-detoxifying agents can thus interact with the oral bioavailability of antibiotics depending on the antibiotic and detoxifying agent, with possible adverse effects on the health of animals and humans.

Agricultural contaminants in amphibian breeding ponds: Occurrence, risk and correlation with agricultural land use.

Anthropogenic pressure such as agricultural pollution globally affects amphibian populations. In this study, a total of 178 different compounds from five agrochemical groups (i.e. antimicrobial drugs residues (ADRs), coccidiostats and anthelmintics, heavy metals, mycotoxins and pesticides) were determined monthly, from March until June 2019 in 26 amphibian breeding ponds in Flanders, Belgium. Furthermore, a possible correlation between the number and concentration of selected contaminants that were found and the percentage of arable land within a 200 m radius was studied. Within each group, the highest detected concentrations were obtained for 4-epioxytetracycline (0.422 µg L-1), levamisole (0.550 µg L-1), zinc (333.1 µg L-1), 3-acetyldeoxynivalenol (0.013 µg L-1), and terbuthylazine (38.7 µg L-1), respectively, with detection frequencies ranging from 1 (i.e. 3-acetyldeoxynivalenol) to 26 (i.e. zinc) out of 26 ponds. Based on reported acute and chronic ecotoxicological endpoints, detected concentrations of bifenthrin, cadmium, copper, cypermethrin, hexachlorobenzene, mercury, terbuthylazine, and zinc pose a substantial ecological risk to aquatic invertebrates such as Daphnia magna and Ceriodaphnia dubia, which both play a role in the food web and potentially in amphibian disease dynamics. Additionally, the detected concentrations of copper were high enough to exert chronic toxicity in the gray treefrog (Hyla versicolor). The number of detected compounds per pond ranged between 0 and 5 (ADRs), 0 - 2 (coccidiostats and anthelmintics), 1 - 7 (heavy metals), 0 - 4 (mycotoxins), and 0 - 12 (pesticides) across the four months. Furthermore, no significant correlation was demonstrated between the number of detected compounds per pond, as well as the detected concentrations of 4-epioxytetracycline, levamisole, copper, zinc, enniatin B and terbuthylazine, and the percentage of arable land within a 200 m radius. For heavy metals and pesticides, the number of compounds per pond varied significantly between months. Conclusively, amphibian breeding ponds in Flanders were frequently contaminated with agrochemicals, yielding concentrations up to the high µg per liter level, regardless of the percentage surrounding arable land, however showing temporal variation for heavy metals and pesticides. This research also identifies potential hazardous substances which may be added to the European watch list (CD 2018/408/EC) in the future.

The influence of modulation of P-glycoprotein and /or cytochrome P450 3A on the pharmacokinetics and pharmacodynamics of orally administered morphine in dogs.

The influence of pretreatment with ketoconazole [cytochrome P450 3A (CYP3A) + P-glycoprotein (P-gp) inhibitor], elacridar (selective P-gp inhibitor) and rifampicin (CYP3A + P-gp inducer) on oral morphine pharmacokinetics and pharmacodynamics was investigated in experimental dogs. Seven beagles were used in a four-way crossover design. Morphine hydrochloride was administered orally (2.5 mg/kg) alone (control group CON) or after pretreatment with ketoconazole (group KETO), elacridar (group ELA) or rifampicin (group RIF). Morphine plasma concentrations were analysed by liquid chromatography-tandem mass spectrometry. Sedation scores (none, mild, moderate or severe) were evaluated subjectively. Dogs were significantly (P < 0.05) more sedated after ketoconazole pretreatment. There were no significant differences between group CON and the other pretreatment groups in pharmacokinetic parameters taking both sexes into account. Sex differences were apparent in some pharmacokinetic parameters of morphine. The area under the plasma concentration time curve (AUC(0-∞) ) was significantly higher, and the total body clearance was significantly lower in male compared to female dogs in all treatment groups. Ketoconazole, rifampicin and elacridar pretreatment had no significant effects on morphine pharmacokinetics, although dogs in the ketoconazole group showed higher sedation scores.

Feasibility of the Ussing chamber technique for the determination of in vitro jejunal permeability of passively absorbed compounds in different animal species.

The aim of this study was to assess the feasibility of the Ussing chamber technique for the determination of the jejunal permeability of passively absorbed, high permeability model compounds (acetaminophen and ketoprofen) in different animal species. Additionally, electrophysiological measurements and histological examination of pre- and post-incubation tissue specimens were performed. Apparent permeability coefficients of turkey and dog jejunum were low and highly variable due to tissue fragility caused by differences in thickness of the remaining intestinal layers after stripping and resulting in severe damage. Pig and horse jejunum were markedly more suitable for permeability determinations and mild signs of deterioration were noticed after 120 min of incubation. Transepithelial electrical resistance and potential difference did not correlate well with the observed tissue damage. From these data, the Ussing chamber technique appears to allow for permeability measurements within a species, but seems unsuitable for interspecies permeability comparison. However, further validation of the method with low permeability compounds and actively transported compounds is needed.

Flow cytometric differentiation of avian leukocytes and analysis of their intracellular cytokine expression.

A flow cytometric method for the identification of chicken blood leukocyte subpopulations and thrombocytes was developed. An anti-chicken CD45 phycoerythrin-labelled antibody was used to separate leukocytes from red blood cell nuclei. Leukocytes and thrombocytes were identified using a combination of their CD45-positivity and their typical side scatter properties. The identity of the CD45-positive cells was confirmed by sorting the subpopulations and subsequent light microscopic evaluation. In these differentiated cell populations, intracellular expression analysis of the proinflammatory cytokines interleukin-1beta and interleukin-6 was subsequently optimized on whole blood after in vitro stimulation with lipopolysaccharide from Escherichia coli strain O127:B8.

Pharmacodynamics of tepoxalin, sodium-salicylate and ketoprofen in an intravenous lipopolysaccharide inflammation model in broiler chickens.

The pharmacodynamic properties of tepoxalin, Na-salicylate and ketoprofen were determined in an intravenous lipopolysaccharide (LPS) inflammation model in broiler chickens. The drugs were administered orally at a dose of 30, 50 and 3 mg/kg, respectively. LPS administration induces an increase in the intracellular expression of interleukin (IL)-1ß and IL-6 and the secreted IL-6 plasma concentration. Furthermore, an elevation in body temperature is noted. Despite pretreatment with a single dose of the drugs and LPS administration on the T(max) of the drug after a second dose, no decrease was seen in systemic IL-6 levels. The intracellular expression of IL-1ß in the heterophils was slightly decreased if LPS was administered in combination with each of the three drugs. Tepoxalin and Na-salicylate administration had no significant effect on the LPS-induced increase in prostaglandin E(2) plasma concentration, in contrast to ketoprofen. None of the three drugs were able to influence the elevation in body temperature after LPS administration. The pharmacokinetic properties of Na-salicylate and ketoprofen were not altered in combination with LPS administration. However, LPS significantly decreased the AUC(0→6 h) of the active metabolite of tepoxalin, RWJ-20142, indicating a perfusion-limited elimination for this molecule.

Multi-class analysis of 46 antimicrobial drug residues in pond water using UHPLC-Orbitrap-HRMS and application to freshwater ponds in Flanders, Belgium.

Increasing anthropogenic pressure and agricultural pollution raises concerns regarding antimicrobial resistance and biodiversity loss in aquatic environments. In order to protect and restore water resources and biodiversity, antimicrobial drug residues should be monitored in all aquatic environments including pond water. Consequently, the objective of this research was to develop and validate a novel multi-residue method for the simultaneous quantification of 46 targeted human and veterinary antimicrobial drugs in pond water. A suitable extraction method based on solid-phase extraction (SPE) was developed, assisted by a fractional factorial design. A broad polarity range of compounds was covered (log P from -4.05 to 4.38), including major representatives of the following classes: sulfonamides, tetracyclines, quinolones, macrolides, lincosamides, nitrofurans, penicillins, cephalosporins, diaminopyrimidines, pleuromutilins and phenicols. All analytes were separated using ultra-high performance liquid chromatography (UHPLC) and detected in full-scan by Orbitrap high resolution mass spectrometry (Orbitrap-HRMS). Good linearity was obtained for all compounds with R2 ≥ 0.993 and goodness-of-fit coefficient (g) ≤ 11.56%. Method detection limits ranged from 10 to 50 ng L-1 and method quantification limits were 50 ng L-1 for all compounds. Acceptable values were obtained for within-day and between-day apparent recoveries (i.e. between 50 and 120%), precision (< 30% and < 45%) and measurement uncertainty (< 50%). Targeted analysis of 18 freshwater ponds throughout Flanders was performed to demonstrate the applicability of the newly developed UHPLC-HRMS method. Overall, 20 antimicrobial drugs were detected with highest concentrations observed for tetracyclines and their transformation products ranging between 51 and 248 ng L-1. Finally, suspect screening was performed suggesting the presence of 14 additional pharmaceuticals including 3 antimicrobial degradation products (e.g. apo-oxytetracycline, amoxicillin penicilloic acid and penilloic acid) and 11 pesticides.

Highly sensitive multi-residue analysis of veterinary drugs including coccidiostats and anthelmintics in pond water using UHPLC-MS/MS: application to freshwater ponds in Flanders, Belgium.

Veterinary drugs, such as coccidiostats and anthelmintics are routinely administered in extensive animal husbandry, finding their way into the aquatic environment through urine and/or feces of treated animals kept outdoors or by the application of contaminated liquid manure on agricultural fields and subsequent mechanisms of surface run-off, leaching and drift. Several of these compounds are known to exert acute and chronic toxicity effects on aquatic organisms, and can lead to changes in biodiversity and ecosystem functioning. The overall objective of this research was to develop, validate and apply a highly sensitive, multi-residue SPE-UHPLC-MS/MS method for the determination of 12 coccidiostats, registered as a feed supplement or veterinary medicine in Europe and three regularly used anthelmintics, in pond water, often functioning as amphibian habitat. Sample extraction was optimized using a fractional factorial resolution design. Pond water filtration efficiency (i.e. 80-118%, ≤25% RSD) and matrix effects (i.e. 72-119%, ≤39% RSD) were evaluated using water from respectively 3 and 20 different ponds in Flanders. By incorporating internal standards, overall results improved and adequate precision values (i.e.≤15%) were obtained according to the EMA guidelines. Acceptable within-run and between-run apparent recoveries, satisfactory precision as well as good linearity were demonstrated according to the CD 2002/657/EC, SANTE/12682/2019 and VICH 49 guidelines, except for robenidine for which the between-day precision was between 21.0 and 34.5%. Sample storage stability studies indicated that storage at 4 °C and analysis performed within 96 hours after sampling was sufficient to avoid loss by degradation for all compounds, excluding robenidine. Values for the limit of detection (LOD) and quantification (LOQ) were in nanograms per liter, which was essential for the environmental application of this novel method. The method was successfully applied on grab water samples from the water surface of 18 different ponds across Flanders, Belgium, detecting amprolium and levamisole at concentrations below the LOQ of 2.5 ng L-1 and at 250.0 ng L-1 or below the LOQ of 250.0 ng L-1, respectively. In conclusion, our newly developed method may provide insights about the contamination status of amphibian breeding ponds.

Characterization of an intravenous lipopolysaccharide inflammation model in broiler chickens.

Intravenous administration of lipopolysaccharide (LPS) from Escherichia coli O127:B8 at a dose of 1,500,000 u/kg body weight evoked a hypothermic response followed by a fever phase in 5-week-old broiler chickens. The hypothermic phase coincided with a severe decrease in blood pressure. We assume that this decrease in blood pressure is, at least partly, responsible for the hypothermic phase of the body temperature curve. LPS administration also caused a decrease in circulating white blood cells. The heterophils were predominantly sequestered in the lungs. In LPS-treated chickens, far more apoptotic leukocytes were present in the circulation, compared with control chickens. The molecular players responsible for the LPS-induced inflammatory response could be TL1A, IL-1beta and IL-6, since a slight increase in their mRNA levels in white blood cells was already seen 1 h after LPS administration. In accordance with these observations, the levels of secreted IL-6 were maximal 3 h after LPS administration. These parameters characterize this LPS-induced inflammation model in broiler chickens.

Evaluation of orally administered valacyclovir in experimentally EHV1-infected ponies.

The purpose of the current study was to investigate the therapeutic efficacy of valacyclovir against EHV1 in a controlled study. Eight naïve Shetland ponies were inoculated with 10(6.5) TCID(50) of the neuropathogenic strain 03P37. Four ponies were treated with valacyclovir at a dosage of 40mg/kg bodyweight, 3 times daily, for 5 (n=2) or 7 (n=2) consecutive days, while the other four ponies served as untreated controls. The treatment regimen started 1h before inoculation. Ponies were monitored daily for clinical signs. At 0, 1, 2, 3, 4, 5, 7, 9, 11, 14, 17 and 21 days post inoculation (d pi), a nasopharyngeal mucus sample was taken to determine viral shedding. At the same time points, blood was collected and peripheral blood mononuclear cells (PBMC) were isolated to determine viremia. During the treatment, blood samples were collected 6 times daily, i.e. just before valacyclovir administration and 1h later, to determine the concentration of acyclovir in plasma. Also a nasopharyngeal swab was taken to measure the acyclovir concentration in nasal secretion. No differences could be noticed between valacyclovir-treated and untreated ponies. The clinical signs, the viral shedding and the viremia were similar in both the groups. Plasma acyclovir concentration could be maintained above the EC(50)-value of EHV1 during 50% of the entire treatment period in valacyclovir-treated ponies. Acyclovir could be detected in nasal swabs at concentrations varying from 50% to 100% of the corresponding plasma concentration. Although sufficiently high acyclovir levels could be reached in plasma and nasal mucus, no effect was seen of the treatment with valacyclovir on clinical signs, viral shedding and viremia of EHV1-infected ponies.