A single LC-MS/MS validated method for tulathromycin quantification in plasma, seminal plasma, and urine to be applied in a pharmacokinetic study in bull

Tulathromycin is a macrolide antibiotic generally used for the treatment of respiratory diseases in cattle and swine. This work proposes an improvement of a previously published LC-MS/MS method for tulathromycin determination in pig serum, here validated in three different bull matrices: plasma, seminal plasma, and urine. The approach is based on a quick protein precipitation with acetonitrile, filtration, and sample dilution before injection, allowing to rapidly process large batches of samples. Analytes separation was obtained using a BEH C18 (50 x 2.1 mm, 1.7 mu m) column, maintained at 40 degrees C with a chromatographic run of 5 min. The method was fully validated over concentration ranges suitable for field levels of tulathromycin found in each matrix (0.01-1 mu g/ml for plasma, 0.05-5 mu g/ml for seminal plasma, and 0.1-10 mu g/m1 for urine), showing good linearity during each day of testing (R-2 always >0.99). Accuracy and precision were within +/- 15% at all QC concentrations in all the three matrices. Furthermore, the use of tulathromycine-d7 as internal standard mitigated the potential impacts of matrix effect. The validated technique was successfully applied to samples collected during a pharmacokinetic study in bulls, allowing to monitor tulathromycin concentrations over time in the three matrices. To our knowledge, this is the first validated approach for LC-MS/MS quantification of tulathromycin in seminal plasma and urine

Enantiospecific pharmacokinetics of intravenous dexmedetomidine in beagles.

The goal of this study was to investigate the pharmacokinetic (PK) behaviour of dexmedetomidine in dogs administered as a pure enantiomer versus as part of a racemic mixture. Eight unmedicated intact purpose-bread beagles were included. Two intravenous treatments of either medetomidine or dexmedetomidine were administered at 10- to 14-day intervals. Atipamezole or saline solution was administered intramuscularly 45 min later. Venous blood samples were collected into EDTA collection tubes, and the quantification of dexmedetomidine and levomedetomidine was performed by chiral LC-MS/MS. All dogs appeared sedated after each treatment without complication. Plasma concentrations of levomedetomidine were measured only in the racemic group and were 51.4% (51.4%-56.1%) lower than dexmedetomidine. Non-compartmental analysis (NCA) was performed for both drugs, while dexmedetomidine data were further described using a population pharmacokinetic approach. A standard two-compartment mammillary model with linear elimination with combined additive and multiplicative error model for residual unexplained variability was established for dexmedetomidine. An exponential model was finally retained to describe inter-individual variability on parameters of clearance (Cl1 ) and central and peripheral volumes of distribution (V1 , V2 ). No effect of occurrence, levomedetomidine or atipamezole could be observed on dexmedetomidine PK parameters. Dexmedetomidine did not undergo significantly different PK when administered alone or as part of the racemic mixture in otherwise unmedicated dogs

Plant Essential Oils as a Tool in the Control of Bovine Mastitis: An Update

Bovine mastitis is a major concern for the dairy cattle community worldwide. Mastitis, subclinical or clinical, can be caused by contagious or environmental pathogens. Costs related to mastitis include direct and indirect losses, leading to global annual losses of USD 35 billion. The primary treatment of mastitis is represented by antibiotics, even if that results in the presence of residues in milk. The overuse and misuse of antibiotics in livestock is contributing to the development of antimicrobial resistance (AMR), resulting in a limited resolution of mastitis treatments, as well as a serious threat for public health. Novel alternatives, like the use of plant essential oils (EOs), are needed to replace antibiotic therapy when facing multidrug-resistant bacteria. This review aims to provide an updated overview of the in vitro and in vivo studies available on EOs and their main components as an antibacterial treatment against a variety of mastitis causing pathogens. There are many in vitro studies, but only several in vivo. Given the promising results of treatments with EOs, further clinical trials are needed

A quick approach for medetomidine enantiomer determination in dog plasma by chiral liquid chromatography–tandem mass spectrometry and application to a pharmacokinetic study

In the present study, a rapid, sensitive and high-throughput liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the determination of medetomidine enantiomers in dog plasma was developed and validated. The separation and individual quantification of chiral compounds can be a tricky task in LC. This is particularly true when target analytes have a relatively small mass, as is the case with medetomidine, a potent and highly specific α2-adrenoceptor agonist widely used in both human and veterinary medicine. The proposed approach is based on a quick liquid-liquid extraction with ethyl acetate and filtration prior to injection. The optimized mobile phase composition allowed to perfectly separate the two enantiomers of medetomidine in a short chromatographic run time, using a cellulose tris(4-methylbenzoate)-based chiral column. A lower limit of quantification of 0.1 ng/mL was reached for both analytes thanks to the high sensitivity and selectivity of MS/MS and the use of racemic medetomidine-d3 as internal standard prevented potential matrix effect. Linearity was satisfying (R2 > 0.99) over the range 0.1-25 ng/mL, as well as within- and between-session accuracy and precision, both always <15%. This method was also applied with success to a series of samples from a pharmacokinetic (PK) study aimed at comparing dex- and levomedetomidine behaviour after administration of the racemic mixture in dogs. The simple extraction procedure, which allows reduced solvent and time consumption without compromising analytical performances, makes this technique a useful tool for this kind of applications even when small animals are involved, due to the small amount of sample required

Deepening the Whole Transcriptomics of Bovine Liver Cells Exposed to AFB1: A Spotlight on Toll-like Receptor 2

Aflatoxin B1 (AFB1) is a food contaminant metabolized mostly in the liver and leading to hepatic damage. Livestock species are differently susceptible to AFB1, but the underlying mechanisms of toxicity have not yet been fully investigated, especially in ruminants. Thus, the aim of the present study was to better characterize the molecular mechanism by which AFB1 exerts hepatotoxicity in cattle. The bovine fetal hepatocyte cell line (BFH12) was exposed for 48 h to three different AFB1 concentrations (0.9 µM, 1.8 µM and 3.6 µM). Whole-transcriptomic changes were measured by RNA-seq analysis, showing significant differences in the expression of genes mainly involved in inflammatory response, oxidative stress, drug metabolism, apoptosis and cancer. As a confirmatory step, post-translational investigations on genes of interest were implemented. Cell death associated with necrosis rather than apoptosis events was noted. As far as the toxicity mechanism is concerned, a molecular pathway linking inflammatory response and oxidative stress was postulated. Toll-Like Receptor 2 (TLR2) activation, consequent to AFB1 exposure, triggers an intracellular signaling cascade involving a kinase (p38β MAPK), which in turn allows the nuclear translocation of the activator protein-1 (AP-1) and NF-κB, finally leading to the release of pro-inflammatory cytokines. Furthermore, a p38β MAPK negative role in cytoprotective genes regulation was postulated. Overall, our investigations improved the actual knowledge on the molecular effects of this worldwide relevant natural toxin in cattle

Discovering the Protective Effects of Quercetin on Aflatoxin B1-Induced Toxicity in Bovine Foetal Hepatocyte-Derived Cells (BFH12)

Aflatoxin B1 (AFB1) induces lipid peroxidation and mortality in bovine foetal hepatocyte-derived cells (BFH12), with underlying transcriptional perturbations associated mainly with cancer, cellular damage, inflammation, bioactivation, and detoxification pathways. In this cell line, curcumin and resveratrol have proven to be effective in mitigating AFB1-induced toxicity. In this paper, we preliminarily assessed the potential anti-AFB1 activity of a natural polyphenol, quercetin (QUE), in BFH12 cells. To this end, we primarily measured QUE cytotoxicity using a WST-1 reagent. Then, we pre-treated the cells with QUE and exposed them to AFB1. The protective role of QUE was evaluated by measuring cytotoxicity, transcriptional changes (RNA-sequencing), lipid peroxidation (malondialdehyde production), and targeted post-transcriptional modifications (NQO1 and CYP3A enzymatic activity). The results demonstrated that QUE, like curcumin and resveratrol, reduced AFB1-induced cytotoxicity and lipid peroxidation and caused larger transcriptional variations than AFB1 alone. Most of the differentially expressed genes were involved in lipid homeostasis, inflammatory and immune processes, and carcinogenesis. As for enzymatic activities, QUE significantly reverted CYP3A variations induced by AFB1, but not those of NQO1. This study provides new knowledge about key molecular mechanisms involved in QUE-mediated protection against AFB1 toxicity and encourages in vivo studies to assess QUE’s bioavailability and beneficial effects on aflatoxicosis

Does Bentonite Cause Cytotoxic and Whole-Transcriptomic Adverse Effects in Enterocytes When Used to Reduce Aflatoxin B1 Exposure?

Aflatoxin B1 (AFB1) is a major food safety concern, threatening the health of humans and animals. Bentonite (BEN) is an aluminosilicate clay used as a feed additive to reduce AFB1 presence in contaminated feedstuff. So far, few studies have characterized BEN toxicity and efficacy in vitro. In this study, cytotoxicity (WST-1 test), the effects on cell permeability (trans-epithelial electrical resistance and lucifer yellow dye incorporation), and transcriptional changes (RNA-seq) caused by BEN, AFB1 and their combination (AFB1 + BEN) were investigated in Caco-2 cells. Up to 0.1 mg/mL, BEN did not affect cell viability and permeability, but it reduced AFB1 cytotoxicity; however, at higher concentrations, BEN was cytotoxic. As to RNA-seq, 0.1 mg/mL BEN did not show effects on cell transcriptome, confirming that the interaction between BEN and AFB1 occurs in the medium. Data from AFB1 and AFB1 + BEN suggested AFB1 provoked most of the transcriptional changes, whereas BEN was preventive. The most interesting AFB1-targeted pathways for which BEN was effective were cell integrity, xenobiotic metabolism and transporters, basal metabolism, inflammation and immune response, p53 biological network, apoptosis and carcinogenesis. To our knowledge, this is the first study assessing the in vitro toxicity and whole-transcriptomic effects of BEN, alone or in the presence of AFB1