Il servizio antiplagio e il Dipartimento di Scienze Mediche Veterinarie della Scuola di Agraria e Medicina Veterinaria dell'Università di Bologna

La Facoltà di Medicina Veterinaria ha aderito alla sperimentazione antiplagio con tutti i corsi di Laurea. Pur trattandosi di sperimentazione, l’analisi con il programma antiplagio è stata resa obbligatoria, fin dall’inizio, per tutte le tesi sia di Laurea sia di Dottorato. L’intervento avrà l’obiettivo di evidenziare la peculiarità della sperimentazione presso la Facoltà di Medicina Veterinaria, il ruolo del referente nei confronti dei colleghi docenti, i vantaggi dell’utilizzo del programma antiplagio e alcune criticità

Plasma and Red Blood Cells Concentration Profiles of Ktamine after Single Intravenous Administration in an Anaesthetic Protocol in Horses

The aim of this study was to describe the concentration profile of ketamine in plasma and red blood cells following an intravenous (IV) bolus in the horse. Ten healthy standardbred horses (two males and height females) 7.7 ± 4.6 (mean value ± SD) years old and weighting 380 ± 21 kg (mean value ± SD) were recruited. The horses were premedicated with acepromazine (0.04 mg·kg-1·IV). Fifteen minutes later they received romifidine (0.08 mg·kg-1·IV), and 5 minutes after they were administered midazolam (0.06 mg·kg-1·IV). Immediately, anaesthesia was induced by ketamine (2.2 mg·kg-1·IV). Venous blood samples were collected at scheduled time points. Plasma and red blood cells (RBCs) concentration of ketamine was assayed using a high performance liquid chromatographic method (HPLC/UV-DAD). The high mean recovery rates, the high sensitivity, the good linearity, suggest a clinical applicability of the analytical method. A bicompartmental model resulted as the most appropriate to describe the ketamine concentration—time profile for both plasma and RBCs. The fitted regression line between ketamine plasma concentrations and RBC concentrations supports the good correlation between ketamine concentrations in plasma and in RBCs. The kinetic parameters of ketamine calculated for RBC are equal or very similar to the plasma ones. The study confirms the kinetic behaviour of ketamine used in the horse as anaesthetic inducers in routine surgery. Finally, the bicompartmental model well describes the ketamine profile also in RBCs, that it is very close to the plasma profile, underlining the great importance of RBCs as blood subcompartment

An LC-MS/MS method for the determination of budesonide and 16α-hydroxyprednisolone in dog plasma

open6noAlthough budesonide is frequently used in veterinary medicine for the treatment of canine respiratory and bowel inflammatory diseases, knowledge is lacking regarding its kinetics in this species. We developed and validated a liquid chromatography–tandem mass spectrometry method for the determination of budesonide and its metabolite 16a-hydroxyprednisolone in dog plasma. The analytes were extracted by solid phase extraction and analysis was performed by high performance liquid chromatography–tandem mass spectrometry, with positive electrospray ionization. This method allows budesonide and one of its main metabolites to be simultaneously quantified in dog plasma at fairly low concentrations. The proposed protocol is very easy and fast to execute, without compromising analytical performances. A small amount (0.5mL) of plasma is required, making this approach suitable for pharmacokinetic studies also in small sized dogs.openGazzotti, Teresa; Barbarossa, Andrea; Zironi, Elisa; Roncada, Paola; Pietra, Marco; Pagliuca, GiampieroGazzotti, Teresa; Barbarossa, Andrea; Zironi, Elisa; Roncada, Paola; Pietra, Marco; Pagliuca, Giampier

Monitoring dynamics in bacterial competition by Imaging Mass Spectrometry

Microbial competition is a mechanism that occurs when two or more microbial species compete for ecological niches to support their survival and growth (Hibbing et al. 2010). Different factors can contribute to the outcome of microbial competition, such as molecules exchanged between the competing organisms for the regulation of cell densities and the initial spatial configuration of the microbe–microbe interaction. Specifically, production of compounds that kill or limit the growth of competing strains or species can promote niche monopolization (Gonzalez et al. 2011). The released compounds include secondary metabolite antibiotics, bacterial peptides or low-molecular-mass organic compounds. In that sense, it is very important to develop tools that could capture metabolic interactions between two or more bacterial populations. Imaging Mass Spectrometry (IMS) enables the visualization of both spatial and temporal production of a huge number of metabolites from a single bacterial species and can observe the effects of multiple microbial signals in an interspecies interaction without using tags or labels (Yang et al. 2009). This technique has the potential to be used for identification of novel metabolites and peptides that were previously undetected by other analytical methods. In this work, a combination of IMS and LC-MS/MS was used to study the competition between Listeria monocytogenes (LM) and Lactococcus lactis (LAC) to investigate the metabolic profile of each bacterium in the interacting microbial colonies. IMS analysis revealed several interesting compounds during interaction of microbial colonies. At least six compounds are uniquely expressed during the interaction between LM and LAC. These results could be useful to setup new molecular strategies in the control of bacterial species for a better food safety

Pharmacokinetic profile and partitioning in red blood cells of romifidine after single intravenous administration in the horse

The aims of this study were to assess the plasma concentrations of romifidine in horses after intravenous injection, to evaluate the red blood cell (RBC) partitioning of the anaesthetic drug, and to improve knowledge regarding its sedative effect in horses describing the pharmacokinetic model. Eight adult Standardbred horses received a single bolus of romifidine at a dosage of 100\ua0\u3bcg/kg. Blood samples (5\ua0mL) were collected immediately before romifidine administration (t0), and at 2, 5, 10, 15, 20, 25, 30, 40, 50, 60, 75, 90, 105, 120, 150 and 180\ua0min after injection. A sedation score was recorded at the same time. The romifidine concentrations in plasma and red blood cells were determined by high performance liquid chromatography (HPLC). The plasma and red blood cell concentrations were correlated with the sedation at each time point. Romifidine produced a satisfactory level of sedation in all animals. The sedation was detectable in all horses for up to 105\ua0min. All the animals returned to normal without any behavioural changes at 180\ua0min. The romifidine concentrations in the red blood cells were significantly higher (P\ua0<\ua00.01) at all time points than those in the plasma. The T1/2\u3b2 was 148.67\ua0\ub1\ua061.59\ua0min and body clearance was 22.55\ua0\ub1\ua06.67\ua0mL/kg per min. The results showed that after a single bolus administration of romifidine, a partitioning in the RBCs was detected

Antioxidant and Ex Vivo Immune System Regulatory Properties of Boswellia serrata Extracts

Boswellia serrata (BS) is an important traditional medicinal plant that currently represents an interesting topic for pharmaceutical research since it possesses several pharmacological properties (e.g., anti-inflammatory, antimicrobial, and antitumour). The safety and versatility of this dietary supplement should allow for its use in numerous pathological conditions; however the quality of the extracts needs to be standardized to increase the clinical success rate resulting from its use. In the present study, different commercially available B. serrata extracts were employed to compare their AKBA content and in vitro antioxidant power. Furthermore, their ability to modulate the immune system regulatory properties was investigated. Our results showed that the AKBA content varied from 3.83 ± 0.10 to 0.03 ± 0.004%, with one sample in which it was not detectable. The highest antioxidant power and phenolic content were shown by the same extract, which also exhibited the highest AKBA concentration. Finally, the BS extracts showed the ability to influence the regulatory and effector T-cell compartments. Our results suggest that frankincense should be further investigated for its promising potentiality to modulate not only inflammation/oxidative stress but also immune dysregulation, but attention should be paid to the composition of the commercial extracts

Food Safety: Secretome of Lactococcus lactis and Listeria monocytogenes in competition.

Listeria monocytogenes (LM) is a foodborne pathogen responsible of listeriosis. In the spreading of this pathology, milk and dairy products are key reservoir for this pathogen1. Food processing represents one of the major steps that could be linked to LM growth. Inhibition of LM growth through competition of Lactococcus lactis (LAC) could represent a solution to this problem. Exoproteome of LM and two different strains of Lactic Acid Bacteria in co-culture have been studied in order to highlight mechanisms of bacterial competition useful to improve food safety. Two different strains of LAC and one strain of LM were cultivated in appropriate medium cultures (BHI), also in competition. Filtrated cultures (SECRETOME) were lyophilized and resuspended for proteomics analysis. Shotgun analysis on each secretome was performed on nano UPLC-MS system. Obtained data reveal, during competition, the higher production by LM of moonlighting protein Enolase and Glucose 6 Phosphate isomerase, of Septation ring formation regulator EzrA, involved into cell replication and the lower secretion of Endopeptidase P60. In parallel, L. lactis produced higher amounts of Secreted 45 kDa protein and switched from lantibiotic Nisin A production to Nisin Z production. In competition with LM, LAC strain investigated produce higher amounts of Secreted 45 kDa protein with peptidoglycan lytic activity and the selective secretion of Nisin Z probably to improve lantibiotic solubility in less acidic environment. Next step will be validation of obtained results in dairy products. These results are of interesting to design new strategies of fighting LM as contaminant in food from animal origin.Work supported by Ministry of Health-CCM “Milano EXPO 2015 Project: Garantire la sicurezza alimentare- Valorizzare le produzioni

Occurrence of Histamine in Commercial Cat Foods under Different Storage Conditions

In fish-based foods, one of the effects of inappropriate storage can be the formation of biogenic amines. Among these, histamine is considered one of the most toxic. The purpose of the present study is to assess the occurrence of histamine in fish-based pet foods, and to evaluate the changes in histamine content during storage at different temperatures. For the analysis, an LC-MS/MS method was used. Fifty-eight pet foods were purchased, and an aliquot of them was analyzed just after opening the packages. Histamine was detected in 12 samples and concentrations ranged from 1.5 to 30.1 mg/kg. The remaining of each positive sample was divided into seven subsamples. One of them was used as a control sample and kept at -20 °C, while the other six were exposed to different environmental conditions. Samples exposed to room temperature showed no significant changes in histamine levels, while samples exposed to high temperatures showed significant increases in histamine content. Finally, samples exposed to refrigerator temperature showed a slight decrease in histamine levels. Under the experimental conditions, the EU limit of 100 mg/kg established for fishery products was never exceeded. These results seem to indicate a low risk of histamine intoxication in cats fed fish-based pet food

Rapid liquid AP-MALDI MS profiling of lipids and proteins from goat and sheep milk for speciation and colostrum analysis

Rapid profiling of the biomolecular components of milk can be useful for food quality assessment and for food fraud detection. Differences in commercial value and availability of milk from specific species are often the reasons for the illicit and fraudulent sale of milk whose species origin is wrongly declared. In this study, a fast, MS-based speciation method is presented to distinguish sheep from goat milk and sheep colostrum at different phases. Using liquid atmospheric pressure (AP)-matrix-assisted laser desorption/ionisation (MALDI) MS, it was possible to classify samples of goat and sheep milk with 100% accuracy in one minute of data acquisition per sample. Moreover, an accuracy of 98% was achieved in classifying pure sheep milk samples and sheep milk samples containing 10% goat milk. Evaluating colostrum quality and postnatal stages represents another possible application of this technology. Classification of sheep colostrum samples that were collected within 6 hours after parturition and 48 hours later was achieved with an accuracy of 84.4%. Our data show that substantial changes in the lipid profile can account for the accurate classification of colostrum collected at the early and late time points. This method applied to the analysis of protein orthologs of different species can, as in this case, allow unequivocal speciation analysis

Alpha1-acid glycoprotein post-translational modifications: a comparative two dimensional electrophoresis based analysis

Alpha1-acid glycoprotein (AGP) is an immunomodulatory protein expressed by hepatocytes in response to the systemic reaction that follows tissue damage caused by inflammation, infection or trauma. A proteomic approach based on two dimensional electrophoresis, immunoblotting and staining of 2DE gels with dyes specific for post-translational modifications (PTMs) such as glycosylation and phosphorylation has been used to evaluate the differential interspecific protein expression of AGP purified from human, bovine and ovine sera. By means of these techniques, several isoforms have been identified in the investigated species: they have been found to change both with regard to the number of isoforms expressed under physiological condition and with regard to the quality of PTMs (i.e. different oligosaccharidic chains, presence/absence of phosphorilations). In particular, it is suggested that bovine serum AGP may have one of the most complex pattern of PTMs among serum proteins of mammals studied so far