Population pharmacokinetics of lisinopril in hypertensive children and adolescents with normal to mildly reduced kidney function.

AIMS: Lisinopril, an angiotensin-converting enzyme inhibitor, is a frequently prescribed antihypertensive drug in the paediatric population, while being used off-label under the age of 6 years in the USA and for all paediatric patients globally. The SAFEPEDRUG project (IWT-130033) investigated lisinopril pharmacokinetics in hypertensive paediatric patients corresponding with the day-to-day clinical population. METHODS: The dose-escalation pilot study included 13 children with primary and secondary hypertension who received oral lisinopril once daily in the morning; doses ranged from 0.05 to 0.2 mg kg-1 . Patients were aged between 1.9 and 17.9 years (median 13.5 years) and weight ranged between 9.62 and 97.2 kg (median 53.2 kg). All data were analysed using Monolix version 2020R1 (Lixoft, France) and R version 3.6.2. RESULTS: A 1-compartment model with first-order absorption and first-order elimination optimally describes the data. Parameter estimates of absorption rate constant (0.075 h-1 [0.062, 0.088], typical value [95% confidence interval]), volume of distribution (31.38 L 70 kg-1 [10.5, 52.3]) and elimination clearance (24.2 L h-1 70 kg-1 [19.5, 28.9]) show good predictive ability. Significant covariate effects include total body weight on elimination clearance, and distribution volume and estimated glomerular filtration rate (eGFR) on elimination clearance. The effects of eGFR on the elimination clearance are optimally described by a linear effect centred around 105 mL min-1  1.73 m-2 . The effects of body weight were implemented using fixed allometric exponents centred around an adult weight of 70 kg. CONCLUSION: Lisinopril dose and regimen adjustments for paediatric patients should include eGFR on top of weight adjustments. An expanded model characterizing the pharmacodynamic effect is required to identify the optimal dose and dosing regimen.

Widespread triazole pesticide use affects infection dynamics of a global amphibian pathogen.

The sixth mass extinction is a consequence of complex interplay between multiple stressors with negative impact on biodiversity. We here examine the interaction between two globally widespread anthropogenic drivers of amphibian declines: the fungal disease chytridiomycosis and antifungal use in agriculture. Field monitoring of 26 amphibian ponds in an agricultural landscape shows widespread occurrence of triazole fungicides in the water column throughout the amphibian breeding season, together with a negative correlation between early season application of epoxiconazole and the prevalence of chytrid infections in aquatic newts. While triazole concentrations in the ponds remained below those that inhibit growth of Batrachochytrium dendrobatidis, they bioaccumulated in the newts' skin up to tenfold, resulting in cutaneous growth-suppressing concentrations. As such, a concentration of epoxiconazole, 10 times below that needed to inhibit fungal growth, prevented chytrid infection in anuran tadpoles. The widespread presence of triazoles may thus alter chytrid dynamics in agricultural landscapes.

No Effect of Acute Balenine Supplementation on Maximal and Submaximal Exercise Performance in Recreational Cyclists.

Carnosine (ß-alanyl-L-histidine) and its methylated analogues anserine and balenine are highly concentrated endogenous dipeptides in mammalian skeletal muscle that are implicated in exercise performance. Balenine has a much better bioavailability and stability in human circulation upon acute ingestion, compared to carnosine and anserine. Therefore, ergogenic effects observed with acute carnosine and anserine supplementation may be even more pronounced with balenine. This study investigated whether acute balenine supplementation improves physical performance in four maximal and submaximal exercise modalities. A total of 20 healthy, active volunteers (14 males; six females) performed cycling sprints, maximal isometric contractions, a 4-km TT and 20-km TT following either preexercise placebo or 10 mg/kg of balenine ingestion. Physical, as well as mental performance, along with acid-base balance and glucose concentration were assessed. Balenine was unable to augment peak power (p = .3553), peak torque (p = .3169), time to complete the 4 km (p = .8566), nor 20 km time trial (p = .2660). None of the performances were correlated with plasma balenine or CN1 enzyme activity. In addition, no effect on pH, bicarbonate, and lactate was observed. Also, the supplement did not affect mental performance. In contrast, glucose remained higher during and after the 20 km time trial following balenine ingestion. In conclusion, these results overall indicate that the functionality of balenine does not fully resemble that of carnosine and anserine, since it was unable to elicit performance improvements with similar and even higher plasma concentrations.

In Vivo Validation of a Reversible Small Molecule-Based Switch for Synthetic Self-Amplifying mRNA Regulation.

Synthetic mRNA therapeutics have the potential to revolutionize healthcare, as they enable patients to produce therapeutic proteins inside their own bodies. However, convenient methods that allow external control over the timing and magnitude of protein production after in vivo delivery of synthetic mRNA are lacking. In this study, we validate the in vivo utility of a synthetic self-amplifying mRNA (RNA replicon) whose expression can be turned off using a genetic switch that responds to oral administration of trimethoprim (TMP), a US Food and Drug Administration (FDA)-approved small-molecule drug. After intramuscular electroporation, the engineered RNA replicon exhibited dose-dependent and reversible expression of its encoded protein upon TMP administration. The TMP serum level needed for maximal downregulation of protein translation was approximately 45-fold below that used in humans for therapeutic purposes. To demonstrate the therapeutic potential of the technology, we injected mice with a TMP-responsive RNA replicon encoding erythropoietin (EPO) and successfully controlled the timing and magnitude of EPO production as well as changes in hematocrit. This work demonstrates the feasibility of controlling mRNA kinetics in vivo, thereby broadly expanding the clinical versatility of mRNA therapeutics.

Development and Validation of a Reliable UHPLC-MS/MS Method for Simultaneous Quantification of Macrocyclic Lactones in Bovine Plasma.

A fast, accurate and reliable ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method was developed for simultaneous quantification of ivermectin (IVER), doramectin (DORA), and moxidectin (MOXI) in bovine plasma. A priority for sample preparation was the eradication of possible infectious diseases to avoid travel restrictions. The sample preparation was based on protein precipitation using 1% formic acid in acetonitrile, followed by Ostro® 96-well plate pass-through sample clean-up. The simple and straightforward procedure, along with the short analysis time, makes the current method unique and suitable for a large set of sample analyses per day for PK studies. Chromatographic separation was performed using an Acquity UPLC HSS-T3 column, with 0.01% acetic acid in water and methanol, on an Acquity H-Class ultra-high performance liquid chromatograph (UHPLC) system. The MS/MS instrument was a Xevo TQ-S® mass spectrometer, operating in the positive electrospray ionization mode and two multiple reaction monitoring (MRM) transitions were monitored per component. The MRM transitions of m/z 897.50 > 753.4 for IVER, m/z 921.70 > 777.40 for DORA and m/z 640.40 > 123.10 for MOXI were used for quantification. The method validation was performed using matrix-matched calibration curves in a concentration range of 1 to 500 ng/mL. Calibration curves fitted a quadratic regression model with 1/x2 weighting (r ≥ 0.998 and GoF ≤ 4.85%). Limits of quantification (LOQ) values of 1 ng/mL were obtained for all the analytes, while the limits of detection (LOD) were 0.02 ng/mL for IVER, 0.03 ng/mL for DORA, and 0.58 ng/mL for MOXI. The results of within-day (RSD < 6.50%) and between-day (RSD < 8.10%) precision and accuracies fell within acceptance ranges. No carry-over and no peak were detected in the UHPLC-MS/MS chromatogram of blank samples showing good specificity of the method. The applicability of the developed method was proved by an analysis of the field PK samples.

Perioperative pharmacokinetics and pharmacodynamics of meloxicam in emus (Dromaius novaehollandiae) of different age groups using nonlinear mixed effect modelling.

Meloxicam is a widely used nonsteroidal anti-inflammatory drug in avian species. However, variability in pharmacokinetic (PK) and pharmacodynamic (PD) parameters in birds warrants species-specific studies for dose and dosing interval optimization. We performed a perioperative PK study of meloxicam (0.5 mg/kg, intravenously) on emus of three different age groups: 3 chicks (5 weeks old, 3.5 kg), 4 juveniles (26 weeks old, 18.8 kg) and 6 adults (66 weeks old, 38.8 kg). A two-compartment population PK model including weight as a significant covariate on clearance and central volume of distribution (V1) best fitted the data. The typical values (20 kg bird) for clearance and V1 were 0.54 L/kg/h and 0.095 L/kg. Both parameters significantly decreased with increasing weight/age. Meloxicam potency and selectivity for COX-1 and COX-2 were measured in whole blood assays (TxB2 production endpoint). Meloxicam was partially selective in emus (IC50 COX-1:COX-2 = 9.1:1). At the current empirical dose (0.5 mg/kg/24 hr), plasma meloxicam concentration is above IC50 of COX-2 for only 2 hr. PK/PD predicted dose required for 80% COX-2 inhibition over 24 hr were 3.4, 1.4 and 0.95 L/kg/day in chicks, juveniles and adult emus, respectively. The safety, therapeutic efficacy and practicality of modifying the daily dose or dose interval should be considered for dose recommendations in emus.

Investigation of age-related differences in toxicokinetic processes of deoxynivalenol and deoxynivalenol-3-glucoside in weaned piglets.

Age-related differences in toxicokinetic processes of deoxynivalenol (DON) and deoxynivalenol-3-glucoside (DON3G) were studied. DON3G [55.7 µg/kg bodyweight (BW)] and an equimolar dose of DON (36 µg/kg BW) were administered to weaned piglets (4 weeks old) by single intravenous and oral administration in a double two-way cross-over design. Systemic and portal blood was sampled at different time points pre- and post-administration and plasma concentrations of DON, DON3G and their metabolites were quantified using validated liquid chromatography-tandem mass spectrometry (LC-MS/MS) and liquid chromatography-high-resolution mass spectrometry (LC-HRMS) methods. Data were processed using tailor-made compartmental toxicokinetic (TK) models to accurately estimate TK parameters. Results were statistically compared to data obtained in a previous study on 11-week-old pigs using identical experimental conditions. Significant age-related differences in intestinal and systemic exposure to both DON and DON3G were noted. Most remarkably, a significant difference was found for the absorbed fraction of DON3G, after presystemic hydrolysis to DON, in weaned piglets compared to 11-week-old piglets (83% vs 16%, respectively), assumed to be mainly attributed to the higher intestinal permeability of weaned piglets. Other differences in TK parameters could be assigned to a higher water/fat body ratio and longer gastrointestinal transit time of weaned piglets. Results may further refine current risk assessment concerning DON and DON3G in animals. Additionally, since piglets possibly serve as a human paediatric surrogate model, results may be extrapolated to human infants.

The role of alanine glyoxylate transaminase-2 (agxt2) in ß-alanine and carnosine metabolism of healthy mice and humans.

PURPOSE: Chronic ß-alanine supplementation leads to increased levels of muscle histidine-containing dipeptides. However, the majority of ingested ß-alanine is, most likely, degraded by two transaminases: GABA-T and AGXT2. In contrast to GABA-T, the in vivo role of AGXT2 with respect to ß-alanine metabolism is unknown. The purpose of the present work is to investigate if AGXT2 is functionally involved in ß-alanine homeostasis. METHODS: Muscle histidine-containing dipeptides levels were determined in AGXT2 overexpressing or knock-out mice and in human subjects with different rs37369 genotypes which is known to affect AGXT2 activity. Further, plasma ß-alanine kinetic was measured and urine was obtained from subjects with different rs37369 genotypes following ingestion of 1400 mg ß-alanine. RESULT: Overexpression of AGXT2 decreased circulating and muscle histidine-containing dipeptides (> 70% decrease; p < 0.05), while AGXT2 KO did not result in altered histidine-containing dipeptides levels. In both models, ß-alanine remained unaffected in the circulation and in muscle (p > 0.05). In humans, the results support the evidence that decreased AGXT2 activity is not associated with altered histidine-containing dipeptides levels (p > 0.05). Additionally, following an acute dose of ß-alanine, no differences in pharmacokinetic response were measured between subjects with different rs37369 genotypes (p > 0.05). Interestingly, urinary ß-alanine excretion was 103% higher in subjects associated with lower AGXT2 activity, compared to subjects associated with normal AGXT2 activity (p < 0.05). CONCLUSION: The data suggest that in vivo, ß-alanine is a substrate of AGXT2; however, its importance in the metabolism of ß-alanine and histidine-containing dipeptides seems small.

The role of roughage provision on the absorption and disposition of the mycotoxin deoxynivalenol and its acetylated derivatives in calves: from field observations to toxicokinetics.

A clinical case in Belgium demonstrated that feeding a feed concentrate containing considerable levels of deoxynivalenol (DON, 1.13 mg/kg feed) induced severe liver failure in 2- to 3-month-old beef calves. Symptoms disappeared by replacing the highly contaminated corn and by stimulating ruminal development via roughage administration. A multi-mycotoxin contamination was demonstrated in feed samples collected at 15 different veal farms in Belgium. DON was most prevalent, contaminating 80% of the roughage samples (mixed straw and maize silage; average concentration in positives: 637 ± 621 µg/kg, max. 1818 µg/kg), and all feed concentrate samples (411 ± 156 µg/kg, max. 693 µg/kg). In order to evaluate the impact of roughage provision and its associated ruminal development on the gastro-intestinal absorption and biodegradation of DON and its acetylated derivatives (3- and 15-ADON) in calves, a toxicokinetic study was performed with two ruminating and two non-ruminating male calves. Animals received in succession a bolus of DON (120 µg/kg bodyweight (BW)), 15-ADON (50 µg/kg BW), and 3-ADON (25 µg/kg) by intravenous (IV) injection or per os (PO) in a cross-over design. The absolute oral bioavailability of DON was much higher in non-ruminating calves (50.7 ± 33.0%) compared to ruminating calves (4.1 ± 4.5%). Immediately following exposure, 3- and 15-ADON were hydrolysed to DON in ruminating calves. DON and its acetylated metabolites were mainly metabolized to DON-3-glucuronide, however, also small amounts of DON-15-glucuronide were detected in urine. DON degradation to deepoxy-DON (DOM-1) was only observed to a relevant extent in ruminating calves. Consequently, toxicity of DON in calves is closely related to roughage provision and the associated stage of ruminal development.

Pharmacokinetics and absolute oral bioavailability of meloxicam in guinea pigs (Cavia porcellus).

OBJECTIVE: To investigate the pharmacokinetics and absolute oral bioavailability of meloxicam in guinea pigs. STUDY DESIGN: Prospective crossover study. ANIMALS: A group of six healthy male Dunkin Hartley guinea pigs. METHODS: A single dose of meloxicam (1.5 mg kg-1) was administered orally and intravenously (IV) to six healthy male guinea pigs. A wash-out period of 48 hours was taken into account between administrations (oral and IV) in the same animal. Blood was sampled through a central venous catheter before administration (t = 0 hours) and at 0.5, 1, 2, 3, 4, 6, 8, 10, 12, 16, 20, 24 and 28 hours post administration. After centrifugation, plasma concentrations of meloxicam were measured by high-performance liquid chromatography with UV detection, and pharmacokinetic parameters were calculated using noncompartmental analysis. RESULTS: Meloxicam in guinea pigs exhibited a moderate absorption rate after oral dosing (time to maximal plasma concentration 3.7 ± 1.7 hours) and maximal plasma concentration was 0.92 ± 0.30 µg mL-1. After IV administration, total body clearance and volume of distribution were 0.13 ± 0.04 and 0.72 ± 0.36 L kg-1, respectively. Terminal half-life was 3.7 ± 0.7 hours and 3.5 ± 1.1 hours after IV and oral administration, respectively. Body extraction ratio was 0.0087 and mean absorption time was 3.8 ± 1.7 hours. The absolute oral bioavailability was 0.54 ± 0.14 in unfasted guinea pigs. CONCLUSIONS AND CLINICAL RELEVANCE: This study reported the pharmacokinetics of meloxicam in guinea pigs. Studies concerning efficacy and safety are the next step towards a rational use of this drug in guinea pigs.

Development of an in Vivo Lipopolysaccharide Inflammation Model to Study the Pharmacodynamics of COX-2 Inhibitors Celecoxib, Mavacoxib, and Meloxicam in Cockatiels (Nymphicus hollandicus).

Nonsteroidal anti-inflammatory drugs (NSAIDs) are used frequently in avian medicine for their antipyretic, analgesic, and anti-inflammatory properties during surgery and for diseases that cause tissue damage and inflammation. NSAIDs inhibit cyclooxygenase (COX) enzymes, which are responsible for the induction of pyresis, pain, and inflammation. In our study, a lipopolysaccharide-induced (LPS) pyresis model was optimized using cockatiels (Nymphicus hollandicus) as subject birds (four males/three females) and validated in two females and one male, characterized by an intravenous bolus injection of LPS (7.5 mg/kg) administered at T0 and T24 (24 hours following the first LPS injection). To demonstrate the feasibility of the model to assess pharmacodynamic (PD) parameters of different NSAIDs, mavacoxib 4 mg/kg (four males/four females), celecoxib 10 mg/kg (four males/four females) and meloxicam 1 mg/kg (four males/four females) were evaluated in the model at dosages used frequently in practice. The PD parameters (body temperature, mentation, posture, preference of location in the cage, and prostaglandin E2 [PGE2] plasma concentrations) were determined for 10 hours following the second LPS injection. At the doses evaluated, mavacoxib and celecoxib significantly reduced LPS-induced hypothermia, but had no clear effects on other clinical signs of illness. In contrast, no effect on hypothermia or clinical appearance was observed in the LPS-challenged cockatiels treated with meloxicam. All three NSAIDs were able to inhibit the increase in LPS-induced PGE2 plasma concentrations, yet the effect was most pronounced in the birds treated with meloxicam. Consequently, the presented model opens perspectives for future dose-effect PD studies to optimize analgesic protocols in cockatiels.

Elevated urinary excretion of free pyridinoline in Friesian horses suggests a breed-specific increase in collagen degradation.

BACKGROUND: Friesian horses are known for their high inbreeding rate resulting in several genetic diseases such as hydrocephaly and dwarfism. This last decade, several studies focused on two other presumed hereditary traits in Friesian horses: megaoesophagus and aortic rupture. The pathogenesis of these diseases remains obscure but an important role of collagen has been hypothesized. The purpose of this study was to examine possible breed-related differences in collagen catabolism. Urinary specimens from Friesian (n = 17, median age 10 years old) and Warmblood horses (n = 17, median age 10 years old) were assessed for mature collagen cross-links, i.e. pyridinoline (PYD) (=hydroxylysylpyridinoline/HP) and deoxypyridinoline (DPD) (lysylpyridinoline /LP). Solid-phase extraction was performed, followed by reversed-phase ion-paired liquid chromatography prior to tandem mass spectrometry (MS/MS) detection. RESULTS: Mean urinary concentrations of free PYD, expressed as fPYD/creatinine ratio, were significantly higher in Friesian horses compared to Warmblood horses (28.5 ± 5.2 versus 22.2 ± 9.6 nmol/mmol, p = 0.02) while mean fDPD/creatinine ratios were similar in both horse breeds (3.0 ± 0.7 versus 4.6 ± 3.7 nmol/mmol, p = 0.09). CONCLUSIONS: Since DPD is considered a specific bone degradation marker and PYD is more widely distributed in connective tissues, the significant elevation in the mean PYD/DPD ratio in Friesian versus Warmblood horses (9.6 ± 1.6 versus 5.7 ± 1.8, p < 0.0001) suggests a soft tissue origin for the increased fPYD levels. Considering that a previous study found no differences in total collagen content between Friesian and Warmblood horses for tendon and aortic tissue, this indicates a higher rate of collagen degradation. The latter might, at least in part, explain the predisposition of Friesians to connective tissue disorders.

Quantification of 8-α-hydroxy-mutilin as marker residue for tiamulin in rabbit tissues by high-performance liquid chromatography-mass spectrometry.

For the first time, a sensitive and specific method was developed and fully validated for the quantification of the EU marker residue of tiamulin, 8-α-hydroxy-mutilin, in rabbit muscle and liver tissues using liquid chromatography combined with positive heated electrospray ionization triple quadrupole mass spectrometry. The mass spectrometer was operated in the selected reaction monitoring (SRM) mode with selection of the [M + H](+) ion in both quadrupoles 1 and 3, resulting in the SRM transition m/z 337.25 > 337.25 for quantification. Chromatography was performed using a Hypersil Gold C18 column using a gradient elution program with water and methanol as mobile phases. The sample preparation procedure for the analysis of 8-α-hydroxy-mutilin in liver and muscle samples consisted of three main steps: (1) extraction of the tissue matrix using 0.1 N hydrochloric acid/acetone (50/50, v/v), (2) hydrolysis of tiamulin and metabolites to 8-α-hydroxy-mutilin in alkaline medium at 45 °C, and (3) liquid-liquid extraction in acidic medium using ethyl acetate. This is the first method presenting fully validated results, encompassing a linearity of 50 to 2,000 µg/kg, within-run and between-run accuracy and precision, limit of quantification (50 µg/kg for both muscle and liver tissues), limit of detection (muscle, 11.9 µg/kg; liver, 20.6 µg/kg), extraction recovery (muscle, 66.2%; liver, 75.5%), signal suppression and enhancement (muscle, 51.7%; liver, 43.3%), carryover, applicability and practicability, and stability during storage and analysis. This novel method is therefore sensitive enough to be used for residue depletion studies of tiamulin in rabbits and for food safety monitoring with respect to MRL compliance of residues.

Use of LC-MS-MS as an alternative to currently available immunoassay methods to quantitate corticosterone in egg yolk and albumen.

Corticosterone (CORT) is the dominant plasma glucocorticoid in birds. There has been increasing interest in the function of CORT in avian egg yolk and in the potential to use CORT concentrations in eggs to quantify stress and to assess the effect of maternal stress on offspring. The concentration of CORT in egg yolk is most frequently assessed using enzyme or radioimmunoassays, alone or in combination with high-performance liquid chromatography. However, the quantification of CORT is frequently hampered by the presence of high concentrations of other steroid hormones which cross-react with the CORT antibody. As an alternative, we developed a sensitive and specific LC-MS-MS method. The sample-preparation procedure consisted of a protein-lipid precipitation step, followed by defatting and clean-up using a C18 SPE column. Chromatography was performed on an Acquity C18 BEH column (50 mm × 2.1 mm i.d., dp: 1.7 µm, run-time: 6 min), using 0.1% formic acid in both water (A) and acetonitrile (B) as mobile phases. The MS-MS instrument was operated in the positive-electrospray-ionization mode. The method was validated in-house according to European Guidelines (linearity, accuracy and precision, limits of quantification and detection, specificity, stability) and the results fell within the accepted ranges. The method was successfully used for the analysis of CORT in yolk and albumen of eggs collected from eight breeding lesser black-backed gulls at a Flemish coastal colony. CORT concentrations were in the range 42.4-166.3 pg g(-1) in albumen and < LOQ (75 pg g(-1))-762.5 pg g(-1) in yolk.

Development of a capillary electrophoretic method for determination of plasma clearance of iohexol in dogs and cats.

Renal function can be monitored by estimation of the glomerular filtration rate (GFR), for example, through measurement of the plasma clearance of a marker that is freely filtrated through the kidney without reabsorption. It has been proposed that iohexol is the most accurate marker for GFR determination in cats and dogs. However, there is a need for a validated capillary electrophoretic method that covers the concentration range for a full curve clearance estimate of iohexol. In the final method, the plasma samples were protein precipitated and the supernatant was analyzed in a background electrolyte containing borate buffer (0.06 m, pH 10.0). The method developed was proved to be linear (concentration range 18- 2900 mg/L) and had a good precision (e.g. 2.3-2.9% at 88 mg/L) and accuracy (e.g. 101-105% at 88 mg/L). Finally, the method was compared with a previously published and validated HPLC-UV method by parallel analysis of clinical plasma samples from dogs and cats administered Omnipaque®. This comparison showed excellent agreement between the two methods and no proportional or systematic error was observed. The proposed method is simple and has a low cost per sample, which makes it applicable for routine analysis.

Pharmacokinetic and pharmacodynamic properties of gamithromycin in turkey poults with respect to Ornithobacterium rhinotracheale.

The macrolide gamithromycin (GAM) has the ability to accumulate in tissues of the respiratory tract. Consequently, GAM might be a suitable antibiotic to treat bacterial respiratory infections in poultry, such as Ornithobacterium rhinotracheale. As O. rhinotracheale infections are common in turkey flocks, the aim of this study was to determine the pharmacokinetic (PK) parameters of GAM in plasma, lung tissue, and pulmonary epithelial lining fluid (PELF) of turkeys and to correlate them with pharmacodynamic (PD) characteristics (PK/PD). The animal experiment was performed with 64 turkeys, which received either a subcutaneous (SC, n=32) or an oral (PO, n=32) bolus of 6 mg GAM/kg body weight (BW). GAM concentrations in plasma, lung tissue, and PELF were measured at different time points post administration (p.a.), and PK characteristics were determined using non-compartmental modeling. The maximum plasma concentration after PO administration was ten-fold lower than after SC injection (0.087 and 0.89 µg/mL, respectively), whereas there was no difference in lung concentrations between both routes of administration. However, lung concentrations at day 1 p.a. were significantly higher than plasma levels for both routes of administration (2.22 and 3.66 µg/g for PO and SC, respectively). Consequently, lung/plasma ratios were high, up to 50 and 80 after PO and SC administration, respectively. GAM could not be detected in PELF, although this might be attributed to the collection method of PELF in birds. The GAM minimum inhibitory concentration (MIC) was determined for 38 O. rhinotracheale strains; MIC50 and MIC90 were 2 and >32 µg/mL, respectively. PK/PD correlation for lung tissue demonstrated that the time above the MIC90 of the susceptible population (2 µg/mL) was 1 day after PO bolus and 3.5 days after SC administration. The area under the curve (AUClast)/MIC ratios for lung tissue after SC and PO administration were 233 and 90, respectively. To conclude, GAM is highly distributed to lung tissue in turkey poults, suggesting that it has the potential to be used to treat respiratory infections such as O. rhinotracheale.

Faecalicoccus acidiformans gen. nov., sp. nov., isolated from the chicken caecum, and reclassification of Streptococcus pleomorphus (Barnes et al. 1977), Eubacterium biforme (Eggerth 1935) and Eubacterium cylindroides (Cato et al. 1974) as Faecalicoccus pleomorphus comb. nov., Holdemanella biformis gen. nov., comb. nov. and Faecalitalea cylindroides gen. nov., comb. nov., respectively, within the family Erysipelotrichaceae.

Strains LMG 27428(T) and LMG 27427 were isolated from the caecal content of a chicken and produced butyric, lactic and formic acids as major metabolic end products. The genomic DNA G+C contents of strains LMG 27428(T) and LMG 27427 were 40.4 and 38.8 mol%. On the basis of 16S rRNA gene sequence similarity, both strains were most closely related to the generically misclassified Streptococcus pleomorphus ATCC 29734(T). Strain LMG 27428(T) could be distinguished from S. pleomorphus ATCC 29734(T) based on production of more lactic acid and less formic acid in M2GSC medium, a higher DNA G+C content and the absence of activities of acid phosphatase and leucine, arginine, leucyl glycine, pyroglutamic acid, glycine and histidine arylamidases, while strain LMG 27428 was biochemically indistinguishable from S. pleomorphus ATCC 29734(T). The novel genus Faecalicoccus gen. nov. within the family Erysipelotrichaceae is proposed to accommodate strains LMG 27428(T) and LMG 27427. Strain LMG 27428(T) ( =DSM 26963(T)) is the type strain of Faecalicoccus acidiformans sp. nov., and strain LMG 27427 ( =DSM 26962) is a strain of Faecalicoccus pleomorphus comb. nov. (type strain LMG 17756(T) =ATCC 29734(T) =DSM 20574(T)). Furthermore, the nearest phylogenetic neighbours of the genus Faecalicoccus are the generically misclassified Eubacterium cylindroides DSM 3983(T) (94.4% 16S rRNA gene sequence similarity to strain LMG 27428(T)) and Eubacterium biforme DSM 3989(T) (92.7% 16S rRNA gene sequence similarity to strain LMG 27428(T)). We present genotypic and phenotypic data that allow the differentiation of each of these taxa and propose to reclassify these generically misnamed species of the genus Eubacterium formally as Faecalitalea cylindroides gen. nov., comb. nov. and Holdemanella biformis gen. nov., comb. nov., respectively. The type strain of Faecalitalea cylindroides is DSM 3983(T) =ATCC 27803(T) =JCM 10261(T) and that of Holdemanella biformis is DSM 3989(T) =ATCC 27806(T) =CCUG 28091(T).

Effect of administration route and dose escalation on plasma and intestinal concentrations of enrofloxacin and ciprofloxacin in broiler chickens.

BACKGROUND: The (mis)use of fluoroquinolones in the fowl industry has led to an alarming incidence of fluoroquinolone resistance in pathogenic as well as commensal bacteria. Next to simply reducing antimicrobial consumption, optimizing dosage regimens can be regarded as a suitable strategy to reduce antimicrobial resistance development without jeopardizing therapy efficacy and outcome. A first step in order to limit antimicrobial resistance is to assess the exposure of the intestinal microbiota to enrofloxacin after different treatment strategies. Therefore, a study was conducted in broiler chickens to assess the effect of route of administration (oral versus intramuscular) and dose escalation (10 and 50 mg/kg body weight) on plasma and intestinal concentrations of enrofloxacin and its main metabolite ciprofloxacin after treatment with enrofloxacin once daily for five consecutive days. Four different parts of the intestinal tract were sampled: ileum, cecum, colon and cloaca. A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed to quantify both analytes in plasma and intestinal content. Sample preparation prior to LC-MS/MS analysis consisted of extraction with ethyl acetate. For intestinal content samples PBS buffer was added before extraction. The supernatant was evaporated to dryness and resuspended in water prior to analysis. RESULTS: The results in plasma and intestinal content demonstrated that biotransformation of enro- to ciprofloxacin in broiler chickens is limited. In general, the intestinal microbiota in cecum and colon is exposed to significant levels of enrofloxacin after conventional treatment (21-130 µg/g). A clear increase of intestinal concentrations was demonstrated after administration of a five-fold higher dose (31-454 µg/g). After intramuscular administration, intestinal concentrations were comparable, except for the higher levels in cloaca due to the complete bioavailability and urinary excretion. CONCLUSIONS: The intestinal microbiota is exposed to high levels of the antimicrobial, after oral as well as parenteral therapy. Furthermore, a dose and time dependent correlation was observed. The impact of the detected intestinal levels on resistance selection in the intestinal microbiota has to be further investigated.

Pharmacokinetic modeling of sulfamethoxazole-trimethoprim and sulfadiazine-trimethoprim combinations in broilers.

Sulfonamides (S) are old bacteriostatic antibiotics which are widely prescribed in combination with trimethoprim (TMP) for the treatment of various diseases in food-producing animals such as poultry. Nowadays, the 1:5 dose ratio of TMP/S used in broilers is a direct transposition of the ratio determined in Human decades ago for TMP/sulfamethoxazole (SMX), aiming to obtain a supposed synergistic plasma concentration ratio of 1:19. However, major pharmacokinetics (PK) differences exist according to the sulfonamide used in the combination. Here, we generated new PK data in broilers after a cross-over design with IV and the oral administration of 2 major sulfonamides, sulfadiazine (SDZ) and SMX, in combination with TMP, and analyzed the data via a population pharmacokinetic (popPK) modeling approach. Results showed that TMP has a greater plasma to tissue distribution than both sulfonamides with a higher volume of distribution (0.51 L/kg for SDZ, 0.62 L/kg for SMX and 3.14 L/kg for TMP). SMX has the highest elimination half-life (2.83 h) followed by SDZ and TMP (2.01 h and 1.49 h, respectively). The oral bioavailability of the 3 molecules was approximately 100%. Bodyweight could explain some of the inter-individual variability in the volume of distribution of SDZ and SMX and the clearance of SDZ and TMP, as heavier broilers have higher typical values. Monte Carlo simulations of a large virtual broiler population (n = 1,000) showed that the targeted plasma ratio of TMP:S of 1:19 was rarely or never reached at the individual level for both combinations at the marketed doses and greatly varies over time and between individuals, questioning the relevance of the 1:5 dose ratio for current formulations of TMP/S.

Actinobacteria as Promising Biocontrol Agents for In Vitro and In Planta Degradation and Detoxification of Zearalenone.

Zearalenone (ZEN) is a prevalent mycotoxin found in grains and grain-derived products, inducing adverse health effects in both animals and humans. The in-field application of microorganisms to degrade and detoxify ZEN is a promising strategy to enhance the safety of food and feed. In this study, we investigated the potential of three actinobacterial strains to degrade and detoxify ZEN in vitro and in planta on wheat ears. The residual ZEN concentration and toxicity in the samples were analysed with UHPLC-MS/MS and a bioluminescence BLYES assay, respectively. Streptomyces rimosus subsp. rimosus LMG19352 could completely degrade and detoxify 5 mg/L ZEN in LB broth within 24 h, along with significant reductions in ZEN concentration both in a minimal medium (MM) and on wheat ears. Additionally, it was the only strain that showed a significant colonisation of these ears. Rhodococcus sp. R25614 exhibited partial but significant degradation in LB broth and MM, whereas Streptomyces sp. LMG16995 degraded and detoxified ZEN in LB broth after 72 h by 39% and 33%, respectively. Although all three actinobacterial strains demonstrated the metabolic capability to degrade and detoxify ZEN in vitro, only S. rimosus subsp. rimosus LMG19352 showed promising potential to mitigate ZEN in planta. This distinction underscores the importance of incorporating in planta screening assays for assessing the potential of mycotoxin-biotransforming microorganisms as biocontrol agents.