OBJECTIVE:To investigate pharmacokinetics (PK) of fentanyl administered by target-controlled infusion (TCI), and to develop a PK model optimized by covariates for TCI in anaesthetized dogs.STUDY DESIGN:Prospective clinical study.ANIMALS:A group of 20 client-owned dogs with spinal pain undergoing anaesthesia for magnetic resonance imaging.METHODS:Fentanyl was administered as an infusion to 20 anaesthetized dogs using a TCI system incorporating a previously described fentanyl two-compartment PK. Arterial blood samples were collected at specific time points during the infusion and over 60 minutes post-infusion for measurement of fentanyl plasma concentrations. The predictive performance of the Sano PK model was assessed by comparing predicted and measured plasma concentrations. A population PK analysis was then performed using a nonlinear mixed-effect modelling approach, allowing inter- and intra-individual variability estimation. Finally, a quantitative stepwise evaluation of the influence of various covariates such as weight, body condition score, size, size-related age, sex and type of premedication on the PK model was considered.RESULTS:Overall predictive performance of the Sano PK set of variables was not clinically acceptable in anaesthetized dogs. Fentanyl PK was best described by a three-compartment model. Weight and sex were found to affect the volume of distribution of the central compartment. Addition of these two covariate/variable associations resulted in a reduction of the objective function value (OFV) from -340.18 to -448.34, and of the median population weighted residual and the median population absolute weighted residual from 16.1% and 38.6% to 3.9% and 20.3%, respectively. Fentanyl infusions at measured concentrations up to 5.4 ng mL-1 in sevoflurane-anaesthetized dogs resulted in stable anaesthesia and smooth recoveries without complications.CONCLUSIONS AND CLINICAL RELEVANCE:A population three-compartment PK model for fentanyl TCI in anaesthetized dogs was developed. Weight and sex have been detected and incorporated as significant covariates.

The use of antimicrobials in agricultural, veterinary and medical practice exerts selective pressure on environmental microbiota, promoting the emergence and spread of antimicrobial resistance (AMR), a global concern for the One Health Initiative Task Force (OHITF). Honeybees have been studied as bioindicators of AMR in the environment, but little is known about beehive products like honey and pollen. The aim of this study was to assess the prevalence of AMR genes (ARGs) in beehive products and investigated their origins. Specifically, possible associations between ARGs, microbiota and other characteristics of different honey and pollen samples, including country of origin, flower type, type of commercial distribution and environmental factors, such as land use, weather and composition of the environment surrounding the beehives were investigated. We found that beehive products harboured ARGs conferring resistance to β-lactams, macrolides, (fluoro)quinolones and polymyxins. Most samples possessed resistance to multiple antimicrobial classes, with honey and pollen showing similar ARG profiles. Even if Lactobacillus and Acinetobacter genera were common in the microbial communities of both honey and pollen, Bacillus, Clostridium, and Bombella defined honey microbiota, while Pseudomonas and Vibrio were enriched in pollen. ErmB and blaTEM-1 co-occurred with Lactobacillus and Fructobacillus, while positive associations between β-lactams and macrolides and anthropogenic environments (i.e. industrial and commercial areas and non-irrigated arable lands) were found. Altogether, our findings suggest that ARGs in honey and pollen might originate from the honeybee foraging environment, and that the beehive products can be used as bioindicators of the AMR environmental contamination.

"WeSocial: Online Learning Community" is a project aiming to provide students with the basic skills in science communication via social media as a useful tool in their future careers and to disseminate the University Department of Comparative Biomedicine and Food Science activities to the general public. The project is based on two main actions: professional training on science communication and social media strategies, and the establishment of an editorial team composed of students supervised by the teaching staff. When the training phase was concluded, official department accounts on Instagram (bca_campus_unipd) and Facebook (BCA_campus_unipd) were opened. Currently, the students' editorial team (SET) oversees publishing a maximum of 3 posts per week, whose content deals with the academic, research, and educational areas of the department seen through the students' eyes. The social media accounts are constantly growing and becoming a "place" for the virtual community of the department. Since students are both "information producers" and the "audience" of the project, they propose and focus on issues particularly important to them. As a result, the department's social media has become a meaningful and relevant experience for students, enhancing their sense of belonging to the departmental and university community life. Moreover, the project is fostering the interaction between students and teaching staff and, thanks to peer communication, is increasing the awareness of department activities especially in the student audience.

A validated method in liquid chromatography tandem mass spectrometry was applied to assess the contamination profile of 17 pyrrolizidine alkaloids and their N-oxide metabolites (PAs/PANOs) in 775 honey samples of different origins, including from local beekeepers (579, LB), large retail and online stores (Italian (LRI) and foreign (LRF) products). Fifty-five percent of the honey samples contained the 17 monitored compounds at below the limit of quantification. The LB samples were characterised by a total mean concentration of PAs/PANOs at 6.7 and 3.3 times lower than those of the LRF and LRI samples, respectively. The PA echimidine characterised mainly honey samples from the large retail stores (75.8 % and 72.5 % of LRI- and LRF-contaminated samples, respectively). The LB samples showed a senecionine-type contamination profile, providing beekeepers clues to the plants responsible for contamination. The results highlighted that the consumption of the local honey samples analysed does not represent a double risk for human health compared to the consumption of products from large retail stores. Careful attention must be paid to honey imported from areas where lycopsamine-type and senecionine-type PAs/PANOs-producing plants are invasive. A machine learning approach could predict the origin of the sample given its contaminant profile at an accuracy of 0.74, which is useful for detecting fraud in the declared honey origin.

The use of bee pollen as a food supplement has increased in recent years as it contains several nutrients and phytochemicals. However, depending on floral composition, bee pollen can be contaminated by pyrrolizidine alkaloids (PAs), PA N-oxides (PANOs) and toxigenic fungi found in plants, which may pose a potential health risk for consumers. Thus, a DNA metabarcoding approach based on internal transcribed spacer 2 (ITS2) region was used to identify the plant sources of 17 PAs/PANOs detected by a validated method in liquid chromatography coupled to mass spectrometry (LC-MS/MS), as well as floral and fungal diversity in 61 bee pollen samples. According to LC-MS/MS analysis, 67% of the samples contained PAs/PANOs with mean concentration of 339 & micro;g/ kg. The contamination pattern was characterised by lycopsamine- and senecionine-type PAs/PANOs. PA/PANOproducing plants were identified in 54% of the PA/PANO-contaminated samples analysed by DNA metabarcoding, which also allowed identifying the overall floral and fungal composition of 56 samples. To evaluate the performance of the molecular approach, a subset of 25 samples was analysed by classical palynology. Palynological analysis partially confirmed the results of DNA metabarcoding, which had a better performance in distinguishing pollens of different genera from Asteraceae (76%) and Brassicaceae (88%). However, the molecular analysis did not identify pollens from Castanea, Eucalyptus, Hedera and Salix, which were abundant in 11 samples according to palynology. On the other hand, the molecular analysis allowed identifying several fungal genera in 33 samples, including the toxigenic fungi Alternaria and Aspergillus, which were positively correlated to the plant genus Hypericum. Despite limitations in identifying some pollen types, these preliminary results suggest that the DNA metabarcoding could be applied in a multidisciplinary approach to give a picture of floral and fungal diversity, which can be sources of natural contaminants in bee pollen and would help to control its safety.

Bee pollen may be contaminated with pyrrolizidine alkaloids (PAs) and their N -oxides (PANOs), which are mainly detected by liquid chromatography coupled to tandem mass spectrometry (LC–MS/MS), even though the use of fast near-infrared (NIR) spectroscopy is an ongoing alternative. Therefore, the main challenge of this study was to assess the feasibility of both a lab-stationary (Foss) and a portable (Polispec) NIR spectrometer in 60 dehydrated bee pollen samples. After an ANOVA-feature selection of the most informative NIR spectral data, canonical discriminant analysis (CDA) was performed to distinguish three quantitative PA/PANO classes (µg/kg): < LOQ (0.4), low; 0.4–400, moderate; > 400, high. According to the LC–MS/MS analysis, 77% of the samples were contaminated with PAs/PANOs and the sum content of the 17 target analytes was higher than 400 µg/kg in 28% of the samples. CDA was carried out on a pool of 18 (Foss) and 22 (Polispec) selected spectral variables and allowed accurate classification of samples from the low class as confirmed by the high values of Matthews correlation coefficient (≥ 0.91) for both NIR spectrometers. Leave-one-out cross-validation highlighted precise recognition of samples characterised by a high PA/PANO content with a low misclassification rate (0.02) as false negatives. The most informative wavelengths were within the < 1000, 1000–1660 and > 2400 nm regions for Foss and > 1500 nm for Polispec that could be associated with cyclic amines, and epoxide chemical structures of PAs/PANOs. In sum, both lab-stationary and portable NIR systems are reliable and fast techniques for detecting PA/PANO contamination in bee pollen.

In humans, the cytochrome P450 3A (CYP3A) subfamily is involved in midazolam (MDZ) biotransformation into 1'- and 4-hydroxy metabolites, and the former serves as a probe for CYP3A catalytic activity. In veterinary species is still crucial to identify enzyme- and species-specific CYP substrates; thus, the aim of this study was to characterize MDZ oxidation in cattle liver. A HPLC-UV method was used to measure 1'- and 4-hydroxy MDZ (1'- and 4-OHMDZ, respectively) formation in cattle liver microsomes and assess the role of CYP3A by an immunoinhibition study. Moreover, MDZ hydroxylation was evaluated in 300 cattle liver samples and results were correlated with testosterone hydroxylation. Formation of both metabolites conformed to a single-enzyme Michaelis-Menten kinetics. Values of Vmax and Km were 0.67 nmol/min/mg protein and 6.16 μM for 4-OHMDZ, and 0.06 nmol/min/mg protein and 10.08 μM for 1'-OHMDZ. An anti-rat CYP3A1 polyclonal antibody inhibited up to 50% and 94% 1'- and 4-OHMDZ formation, respectively. MDZ oxidation in liver microsomes was poorly correlated with testosterone hydroxylation. In conclusion, cattle metabolized MDZ to 1'-OHMDZ and 4-OHMDZ. The immunoinhibition results indicated a major contribution of CYP3As to 4-OHMDZ formation and the involvement of other CYPs in 1'-OHMDZ production, paving the way for further investigations.

Toxic pyrrolizidine alkaloids (PAs) and their N-oxides (PANOs) can be present in bee pollen depending on the plants visited by bees. A liquid chromatography-mass spectrometry (LC-MS/MS) method was developed and validated to monitor 17 PAs/PANOs in 44 bee pollens. The CIE-L∗a∗b∗ colour coordinates with the specular component either included or excluded were recorded in pellets and ground aliquots. Lightness (L∗) and yellowness (b∗) of ground bee pollen were significantly correlated to PAs/PANOs content. The L∗ and b∗ cut-offs sorted by a receiver operating characteristic analysis to predict PAs/PANOs presence showed a significant increase in the relative risk to detect amounts higher than 84 μg kg−1. Two supervised canonical discriminant analyses confirmed that pollen without PAs could be distinguished from those containing PAs/PANOs. The data suggest that instrumental colour coupled with supervised models could be used as a screening test for PAs/PANOs in bee pollen, before the confirmatory LC-MS/MS analysis.

Due to its chemical properties, honey does not foster the growth of microorganisms, however it may contain a rich microbial community, including viable, stressed, and not viable microbes. In order to characterize honey microbiota focusing on the difference between products from beekeepers and large retail in the present study a culture-independent approach based on DNA metabarcoding was applied. Honey samples were collected from Local Beekeepers (LB) and Market sales (M) during four years with the aim to investigate the microbiological quality in the honey market. Extraction and amplification of DNA from honey samples showed reduced efficiency with increasing age of honey, with the loss of 50-80% of samples four years old (2014). For this reason, only samples of similar age were compared and the analysis of microbial communities focused on year 2017, for a total of 75 samples. Differences in alpha and beta-diversity were evidenced comparing microbial communities between LB and M samples. In particular, contaminant bacteria dominated the microbiota in M samples while LB samples were enriched in Lactic Acid Bacteria (LAB) that cannot be isolated with culture-dependent approaches.

Anaesthetics administered during C-section (CS) can cross the placenta and the foetal blood-brain barrier contributing to distress up to neonatal mortality. Therefore, to prevent neonatal risks, sedatives and analgesics are not commonly administered to the bitch until all pups are delivered. This study aims to evaluate the effect of a new anaesthetic and analgesic protocol for elective CS in dogs, focused on both maternal and neonatal wellbeing. General anaesthesia was induced by a combination of propofol (PPF) and dexmedetomidine (DEX) and maintained with isoflurane. DEX was added to PPF in order to provide analgesia and to reduce PPF dose. Propofol and DEX concentrations in maternal blood, amniotic fluid, and placenta were correlated to maternal and neonatal parameters. Maternal pain score was assessed with Glasgow Composite Measure Pain Scale short-form. Nine healthy purebred dogs scheduled for elective CS delivered 54 pups. The 77.8% of pups were vigorous at birth and assigned to the highest Apgar score (AS). The lowest AS was recorded in pups from mothers receiving additional doses of PPF (p < 0.001). Apgar scores improved with the increase in time between induction and pups' extraction, starting from 30 min after induction (p < 0.01). This study could contribute to clarify the controversy about the optimal extraction's time of pups after induction i.e. the best time between PPF administration and birth. No bitch showed post-operative pain or required additional analgesic doses based on their pain score. Maternal blood PPF and DEX, as well as placental PPF concentrations, decreased over time (p < 0.01). Conversely, placental DEX was fair uniformly detected in littermate pups. Both PPF and DEX were not detectable in amniotic fluid. Placenta resulted an effective barrier against foetal DEX exposure, making this protocol safe, analgesic and advisable for elective CS in dogs.

In this work the feasibility of near infrared spectroscopy was evaluated combined with chemometric approaches, as a tool for the botanical origin prediction of 119 honey samples. Four varieties related to polyfloral, acacia, chestnut, and linden were first characterized by their physical–chemical parameters and then analyzed in triplicate using a near infrared spectrophotometer equipped with an optical path gold reflector. Three different classifiers were built on distinct multivariate and machine learning approaches for honey botanical classification. A partial least squares discriminant analysis was used as a first approach to build a predictive model for honey classification. Spectra pretreatments named autoscale, standard normal variate, detrending, first derivative, and smoothing were applied for the reduction of scattering related to the presence of particle size, like glucose crystals. The values of the descriptive statistics of the partial least squares discriminant analysis model allowed a sufficient floral group prediction for the acacia and polyfloral honeys but not in the cases of chestnut and linden. The second classifier, based on a support vector machine, allowed a better classification of acacia and polyfloral and also achieved the classification of chestnut. The linden samples instead remained unclassified. A further investigation, aimed to improve the botanical discrimination, exploited a feature selection algorithm named Boruta, which assigned a pool of 39 informative averaged near infrared spectral variables on which a canonical discriminant analysis was assessed. The canonical discriminant analysis accounted a better separation of samples according to the botanical origin than the partial least squares discriminant analysis. The approach used has permitted to achieve a complete authentication of the acacia honeys but not a precise segregation of polyfloral ones. The comparison between the variables important in projection and the Boruta pool showed that the informative wavelengths are partially shared especially in the middle and far band of the near infrared spectral range.

Cytochrome P450 3A is the most important CYP subfamily in humans, and CYP3A4/CYP3A5 genetic variants contribute to inter-individual variability in drug metabolism. However, no information is available for bovine CYP3A (bCYP3A). Here we described bCYP3A missense single nucleotide variants (SNVs) and evaluated their functional effects. CYP3A28, CYP3A38 and CYP3A48 missense SNVs were identified in 300 bulls of Piedmontese breed through targeted sequencing. Wild-type and mutant bCYP3A cDNAs were cloned and expressed in V79 cells. CYP3A-dependent oxidative metabolism of testosterone (TST) and nifedipine (NIF) was assessed by LC-MS/MS. Finally, SNVs functional impact on TST hydroxylation was measured ex vivo in liver microsomes from individually genotyped animals. Thirteen missense SNVs were identified and validated. Five variants showed differences in CYP3A catalytic activity: three CYP3A28 SNVs reduced TST 6β-hydroxylation; one CYP3A38 variant increased TST 16β-hydroxylation, while a CYP3A48 SNV showed enhanced NIF oxidation. Individuals homozygous for rs384467435 SNV showed a reduced TST 6β-hydroxylation. Molecular modelling showed that most of SNVs were distal to CYP3A active site, suggesting indirect effects on the catalytic activity. Collectively, these findings demonstrate the importance of pharmacogenetics studies in veterinary species and suggest bCYP3A genotype variation might affect the fate of xenobiotics in food-producing species such as cattle.

We evaluated the effects of pre-processing thermal treatments on the physicochemical, colour and near-infrared (NIR) spectral data of 30 honey samples. The trial was settled as a bi-factorial experimental design that considered nine experimental groups according to the fixed effects of heating treatment and honey phase: none, mild (39 °C for 30′) and high heating (55 °C for 24 h) per crystallised, bi-phase and liquid honey samples. Increasing temperatures significantly modified moisture, hydroxymethylfurfural content and lightness. The multivariate classifier models showed that NIR data of warmed crystallised and bi-phase honeys were significantly different from that of the untreated ones, while they sorted a similar assignment for all the liquid samples. The support vector machine model confirmed that the highest tested temperature represented a bias in the informative feature of NIR data, if they would be used in further analytical assessment of the intrinsic qualities of crystallised or bi-phase honey.

The interest in soy isoflavones, especially genistein and daidzein, has increased since scientific data showing a wide range of biological activities of these phytoestrogens. The aim of the present research was to develop and validate a new analytical method for the identification and quantification of isoflavones for the first time in rainbow trout muscle, by the QuEChERS methodology, followed by liquid chromatography coupled with electrospray ionisation mass spectrometry in positive ion (LC-ESI-MS) analysis. The analytes monitored and quantified were Daidzein, Daidzin, Genistein, Genistin, Glycitein and Glycitin. The method was validated according to the criteria of European Commission Decision 2002/657/EC, and the parameters evaluated included linearity, precision, matrix effect, limit of detection and quantification, absolute recovery and robustness. The limit of quantification was fixed at 0.2 lig kg(-1) for all isoflavones, and the absolute recovery ranged from 51.8% to 103.3%. The method was applied to 26 real samples of the rainbow trout muscle, and the correlation between the presence of isoflavones, both free and conjugated forms, in muscle and diet with soybeans and soy derivatives was established. This method is suitable for more accurately determining the human exposure to dietary isoflavones from products of animal origin.

In this study, the effects of both continuous and alternate exposure to 2 mg L-1 of flumequine (FLU) on survival, growth and reproduction of Daphnia magna were evaluated over four generations. Mortality was the most evident effect, with an average mortality rate of 23 +/- 14% across generations. Individuals destined to succumb were identifiable well in advance through their discolouration and lack of development, and limited or zero reproductive capacity. Inhibition of reproduction in surviving mothers varied across the four generations (14.3 +/- 17%) without an apparent correlation with the duration of exposure over generations. Significant reproductive inhibition was observed in the generation that followed three non-exposed generations (the fourth generation), pointing to a transgenerational toxicity of FLU. In another experiment, in vitro exposure of 72 D. magna embryos to 2 mg L-1 FLU caused 14% mortality (versus 7% in the control). Among the 62 individuals that hatched alive, six showed birth defects and only one was able to survive the next few days. The other, apparently healthy newborns were randomly assigned to two groups and submitted to a reproduction test, either in the absence or in the presence of 2 mg L-1 FLU. A high mortality rate and/or strongly significantly inhibited reproduction were detected in both groups. As with previously run analogous tests with enrofloxacin, the multi generational and embryonic tests showed a clear disruption to this crustacean population which would not be evidenced by the standard official acute and chronic tests. This indicates the necessity of taking a different and more comprehensive approach to the evaluation of substances having an inherent ability to interact with genetic material.

Pyrrolizidine alkaloids (PAs) are natural toxins, supposed to be converted to hepatotoxic metabolites in the liver of mammalians, and preventing the uptake of PAs is the only way to reduce the attendant health risks. A liquid chromatography coupled to mass spectrometry (LC-MS/MS) method was developed according to the Decision 2002/657/EC, to evaluate the contamination of Veneto honey by the following pyrrolizidine alkaloids: echimidine, heliotrine, lycopsamine, retrorsine, senecionine, and seneciphylline, as well as their N-oxides (pyrrolizidine alkaloid N-oxides (PANOs)). Before the solid-phase extraction, a reduction step with zinc dust in the presence of sulfuric acid was carried out for the reduction of PANOs to PAs in honey samples. For all of the analytes, the mean recovery ranged between 82.70 and 104.16 % and the limit of quantification (LOQ) was 0.25 μg kg−1, fully satisfying the LOQ of 1 μg kg−1 as requested by EFSA (2012). The validated method was applied for the analysis of 60 Veneto samples produced from local beekeepers. Among the samples, 17 % were positive for the PAs monitored, and their total PA contents ranged from 0.6 to 17.6 μg kg−1. In all of the samples, the contribution from the PANOs appeared to be negligible. The total PA contents found in the Veneto honey samples never exceeded the limit of 0.42 μg per day for an individual weighing 60 kg, established by the German Federal Institute of Risk Assessment (BfR) in 2011. These preliminary results provide useful advice to beekeepers for finding the most suitable location for beehives to achieve a substantial reduction of the PAs in honey.

Introduction - Dexamethasone (DEX), betamethasone (BETA) and prednisolone (PREDN) are synthetic corticosteroids authorized in cattle breeding for the treatment of metabolic disorders and inflammatory diseases but they are illegally used, at lower doses as growth promoters, to improve productive performances. To safeguard the health of the consumer, the EU (Directive 2008/97 /EC) has banned this practice(1).Aim - During 2007, a surveillance plan was conducted in several slaughterhouses of the Veneto Region and 300 cattle were monitored for the presence of illicit treatments with corticosteroids.Materials and methods - Liver, thymus and urine from bladder, were collect to detect, through chemical and histological examination, the presence of corticosteroids treatment. Based on the results of the histological analyses on thymus samples, the animals were classified into 23 Suspected/Doubts and 16 Negative. Liver and urine samples were analyze for the presence of residues of corticosteroids by a multi-residue method in liquid chromatography coupled with mass spectrometry (LC-MS/MS), able to identify and quantify the presence of DEX, BETA and PREDN.Results - The confirmatory method adopted, revealed DEX presence in 11 samples of bovine liver, 10 from the group "suspect" by histological examination and only one sample from the group of negative ones. In 6 liver samples DEX concentrations were above MRL (2 ng/g) and 4 urine samples collected from the same animals revealed DEX detectable concentrations.From published data on DEX elimination rate and from liver and urine results of the monitoring plan, it can be stated that the treatment with DEX in positive animals continued until a few days before slaughtering.Conclusions - The histological examination can be used as first screening of animals illegally treated with DEX. The results obtained confirm the risk for the consumer of foodstuffs of animal origin from cattle, treated with corticosteroids until slaughtering time.