Illicit Treatment Research Articles

Occurrence of resorcyclic acid lactones in porcine urine: discrimination between illegal use and contamination

Zeranol (α-zearalanol, α-ZAL), is a resorcyclic acid lactone (RAL). Its administration to farm animals to improve meat production has been prohibited in the European Union due to the potential risk to human health. However, it has been demonstrated that α-ZAL may be present in livestock animals due to Fusarium fungi that produce fusarium acid lactones contamination in feed. The fungi produce a small amount of zearalenone (ZEN), which is metabolized to zeranol. The potential endogenous origin of α-ZAL makes it difficult to correlate positive samples to a potential illicit treatment with α-ZAL. We present two experimental studies that investigated the origin of natural and synthetic RALs in porcine urine. Urine samples from pigs that were either fed with ZEN-contaminated feed or administered α-ZAL by injection were analyzed by liquid chromatography coupled to tandem mass spectrometry, with the method validated according to Commission Implementing Regulation (EU) 2021/808. The data show that although the concentration of α-ZAL in the ZEN feed-contaminated samples is significantly lower than in the illicit administration samples, α-ZAL can occur in porcine urine via natural metabolism. Additionally, the feasibility of using the ratio of forbidden/fusarium RALs in porcine urine as a reliable biomarker for illicit treatment with α-ZAL administration was evaluated for the first time. This study demonstrated that the obtained ratio in the contaminated ZEN feed study was close to 1, while in the illegally administered α-ZAL samples the ratio is always higher than 1 (up to 135). Therefore, this study proves that the ratio criteria (already used when a forbidden RAL is detected in bovine urine) may also be used for porcine urine.

Identification of Illicit Conservation Treatments in Fresh Fish by Micro-Raman Spectroscopy and Chemometric Methods.

In the field of food control for fresh products, the identification of foods subjected to illicit conservation treatments to extend their shelf life is fundamental. Fresh fish products are particularly subjected to this type of fraud due to their high commercial value and the fact that they often have to be transported over a long distance, keeping their organoleptic characteristics unaltered. Treatments of this type involve, e.g., the bleaching of the meat and/or the momentary abatement of the microbial load, while the degradation process continues. It is therefore important to find rapid methods that allow the identification of illicit treatments. The study presented here was performed on 24 sea bass samples divided into four groups: 12 controls (stored on ice in the fridge for 3 or 24 h), and 12 treated with a Cafodos-like solution for 3 or 24 h. Muscle and skin samples were then characterized using micro-Raman spectroscopy. The data were pre-processed by smoothing and taking the first derivative and then PLS-DA models were built to identify short- and long- term effects on the fish's muscle and skin. All the models provided the perfect classification of the samples both in fitting and cross-validation and an analysis of the bands responsible for the effects was also reported. To the best of the authors' knowledge, this is the first time Raman spectroscopy has been applied for the identification of a Cafodos-like illicit treatment, focusing on both fish muscle and skin evaluation. The procedure could pave the way for a future application directly on the market through the use of a portable device.

Transcriptional Biomarkers and Immunohistochemistry for Detection of Illicit Dexamethasone Administration in Veal Calves

Corticosteroids such as Dexamethasone (DEX) are commonly licensed for therapy in meat animals due to their known pharmacological properties. However, their misuse aimed to achieve anabolic effects is often found by National Residues Control Plans. The setup of a complementary “biomarker based” methods to unveil such illicit practices is encouraged by current European legislation. In this study, the combined use of molecular and histological quantitative techniques was applied on formalin fixed paraffin embedded (FFPE) muscle samples to assess the effects of illicit DEX treatment on veal calves. A PCR array, including 28 transcriptional biomarkers related to DEX exposure, was combined with a histochemical analysis of muscle fiber. An analysis based on unsupervised (PCA) and supervised (PLS-DA and Kohonen’s SOM) methods, was applied in order to define multivariate models able to classify animals suspected of illicit treatment by DEX. According to the conventional univariate approach, a not-significant reduction in type I fibres was recorded in the DEX-treated group, and only 12 out of 28 targeted genes maintained their expected differential expression, confirming the technical limitations of a quantitative analysis on FFPE samples. However, the multivariate models developed highlighted the possibility to establish complementary screening strategies, particularly when based on transcriptional biomarkers characterised by low expression profiles.

Validation of serum paraoxonase/arylesterase 1 (PON1) as a protein marker of illicit dexamethasone treatment in veal calves

The illicit use of dexamethasone and other glucocorticoids for cattle fattening in livestock production has been widely described; evidence for illegal treatments can be obtained by direct or indirect detection. In our previous study, we applied two-dimensional electrophoresis (2DE) to identify plasma protein markers of dexamethasone administration in veal calves. Comparison of 2DE maps obtained from blood samples before and after treatment showed the disappearance of two protein spots identified as serum paraoxonase/arylesterase 1 precursor (PON1). In the present study, we validated PON1 as a marker by analysing a larger number of samples treated with dexamethasone for illicit use. Analysis of samples from experimental treatment with other glucocorticoids, androgens and oestrogens confirmed that their influence on PON1 could be excluded. The specificity of the PON1 protein marker was verified on expected negative field samples to exclude interfering factors. However, there is poor statistical evidence to support a significant association between the outcome of PON1 and the considered variables. The results on field samples were compared with histological examination of the thymus as a biomarker of corticosteroid treatment monitored in the Italian histological plan for the control of growth promoters in animals. Two suspect cases were identified from two Piedmont farms where other animals had tested positive at histological examination. In conclusion, the absence of PON1 in the plasma of veal calves can indirectly reveal illicit dexamethasone treatment in individual animals and so identify suspect farms for further investigation. It is effective in a period ranging from 3 to about 10 days from illicit treatment, covering a time span that goes beyond the limits of official chemical controls and preceding histological controls on the thymus of slaughtered animals. PON1 detection in plasma can be coupled with other tests to identify illegal dexamethasone use on veal calf farms.

Modeling the Dynamics of Heroin and Illicit Opioid Use Disorder, Treatment, and Recovery

Opioid use disorder (OUD) has become a serious leading health issue in the USA leading to addiction, disability, or death by overdose. Research has shown that OUD can lead to a chronic lifelong disorder with greater risk for relapse and accidental overdose deaths. While the prescription opioid epidemic is a relatively new phenomenon, illicit opioid use via heroin has been around for decades. Recently, additional illicit opioids such as fentanyl have become increasingly available and problematic. We propose a mathematical model that focuses on illicit OUD and includes a class for recovered users but allows for individuals to either remain in or relapse back to the illicit OUD class. Therefore, in our model, individuals may cycle in and out of three different classes: illicit OUD, treatment, and recovered. We additionally include a treatment function with saturation, as it has been shown there is limited accessibility to specialty treatment facilities. We used 2002–2019 SAMHSA and CDC data for the US population, scaled to a medium-sized city, to obtain parameter estimates for the specific case of heroin. We found that the overdose death rate has been increasing linearly since around 2011, likely due to the increased presence of fentanyl in the heroin supply. Extrapolation of this overdose death rate, together with the obtained parameter estimates, predict that by 2038 no endemic equilibrium will exist and the only stable equilibrium will correspond to the absence of heroin use disorder in the population. There is a range of parameter values that will give rise to a backward bifurcation above a critical saturation of treatment availability. We show this for a range of overdose death rate values, thus illustrating the critical role played by the availability of specialty treatment facilities. Sensitivity analysis consistently shows the significant role of people entering treatment on their own accord, which suggests the importance of removing two of the most prevalent SAMHSA-determined reasons that individuals do not enter treatment: financial constraints and the stigma of seeking treatment for heroin use disorder.

Exploring the effectiveness of dextroamphetamine for the treatment of stimulant use disorder:\xa0a qualitative study\xa0with patients receiving injectable opioid agonist treatment

BackgroundA high proportion of people receiving both oral and injectable opioid agonist treatment report concurrent use of stimulants (i.e. cocaine and or amphetamines), which has been associated with higher rates of continued illicit opioid use and treatment dropout. A recent randomized controlled trial demonstrated the effectiveness of dextroamphetamine (a prescribed stimulant) at reducing craving for and use of cocaine among patients receiving injectable opioid agonist treatment. Following this evidence, dextroamphetamine has been prescribed to patients with stimulant use disorder at a clinic in Vancouver. This study investigates perceptions of the effectiveness of dextroamphetamine from the perspective of these patients.MethodsData were collected using small focus groups and one-on-one interviews with patients who were currently or formerly receiving dextroamphetamine (n = 20). Thematic analysis was conducted using an iterative approach, moving between data collection and analysis to search for patterns in the data across transcripts. This process led to the defining and naming of three central themes responding to the research question.ResultsParticipants reported a range of stimulant use types, including cocaine (n = 8), methamphetamine (n = 8), or both (n = 4). Three central themes were identified as relating to participants’ perceptions of the effectiveness of the medication: 1) achieving a substitution effect (i.e. extent to which dextroamphetamine provided a substitution for the effect they received from use of illicit stimulants); 2) Reaching a preferred dose (i.e. speed of titration and effect of the dose received); and 3) Ease of medication access (i.e. preference for take home doses (i.e. carries) vs. medication integrated into care at the clinic).ConclusionIn the context of continued investigation of pharmacological treatments for stimulant use disorder, the present study has highlighted how the study of clinical outcomes could be extended to account for factors that contribute to perceptions of effectiveness from the perspective of patients. In practice, elements of treatment delivery (e.g. dosing and dispensation protocols) can be adjusted to allow for various scenarios (e.g. on site vs. take home dosing) by which dextroamphetamine and other pharmacological stimulants could be implemented to provide “effective” treatment for people with a wide range of treatment goals and needs.

Undeclared (Poly)phosphates Detection in Food of Animal Origin as a Potential Tool toward Fraud Prevention

(Poly)phosphates are approved as water-preserving and emulsifying agents that improve the appearance and consistency of many food products. The labelling of added (poly)phosphates is essential for protecting vulnerable population groups and to prevent unfair trade practices resulting in economic fraud. The problems with (poly)phosphates’ utilisation concerns both analytical and legislative issues, such as: (1) their straightforward detection; (2) excessive addition altering freshness perception and misleading consumers; (3) uncontrolled usage increasing foodstuff weight; (4) application in products where they are not permitted; and (5) no indication on the label. Bearing all these issues in mind, the main purpose of this study was the quantification and screening of the (poly)phosphates profile in meat, marine and dairy products (160 samples), of which 43 were without declared (poly)phosphate treatment. Analysis was completed by high-performance ion-exchange chromatography either with conductometric detection or coupled to Q-Exactive Orbitrap high-resolution mass spectrometry. Although the (poly)phosphates profiles varied greatly according to species and processing type, the following criteria for detection of illicit treatment were established: high orthophosphate level, quantified short-chain (poly)phosphate anions and the presence of long-chain forms. In conclusion, the instrumental platforms used in this study can be recommended to inspection bodies as reliable methods for the detection of food adulteration with (poly)phosphates.

Profiling of transcriptional biomarkers in FFPE liver samples: PLS-DA applications for detection of illicit administration of sex steroids and clenbuterol in veal calves

Administering growth promoters to meat-producing animals is strictly regulated within the European Community to preserve consumers’ safety. Nevertheless, illicit misuse of steroids and other veterinary drugs to increase animal production has still been found by National Residues Control Plans (NRCPs), mainly based on expensive targeted multi-class/multi-residue analytical methods, used for confirming quantification analysis of these residues in tissues and biological fluids. The setup of complementary novel diagnostic methods, based on indirect biomarkers of exposure to anabolic substances is, therefore, needed to update current tests available in NRCPs at the screening level, where cheaper, sensitive and untargeted methods are required. Biological effects of illicit treatment transcriptomic analysis has reached a good grade of standardization, identified by microarray and RNAseq technologies of large panels of differentially expressed gene targets (DEG) related to abuse of different classes of anabolic molecules (e.g. sex steroids, thyreostats, β2-agonists, glucocorticoids, etc.). The aim of the work was to identify a minimum set of molecular markers able to detect illicit administration of two of the most recurrent classes of growth promoters (sex steroids and B2-agonists) on formalin-fixed paraffin-embedded (FFPE) tissues, being a potentially straightforward sampling strategy at the slaughterhouse level. A PCR array to allow quantitative profiling of 48 genetic targets was, therefore, developed on FFPE liver samples from veal calves. Conventional univariate and alternative approaches based on multivariate classification methods were considered for gene expression profiling of 92 samples collected from different animal trials, where effects of nandrolone, estradiol, their combination, and clenbuterol were studied.The developed Partial Least Squares – Discriminant Analysis (PLS-DA) models show the successful setup of a novel screening strategy based on multiple transcriptional biomarkers, able to expand tested sample sizes, and consequently strengthen current NRCPs.

Outcomes in chest feminization patients with a history of illicit hormone use and silicone injections.

Transfeminine patients facing barriers to gender confirming surgery sometimes pursue illicit feminization procedures such as "street" hormones and silicone injections. These procedures often yield complications and disfigurement, making surgeons and insurance companies wary of secondary breast reconstruction. This clinical series assesses illicit treatment among our transfeminine breast surgery patients and describes the presentation, surgical approaches, and outcomes of each patient. A retrospective review of transfeminine patients who underwent chest feminization surgery with a single provider between March 2018 and March 2020 identified patients with a history of illicit hormones or silicone injections. Demographics, comorbidities, preoperative diagnoses and symptoms, surgical details, and postoperative outcomes were collected. Patients were contacted for a follow-up survey concerning outcomes and satisfaction. Of the 41 transfeminine patients reviewed, eight (19.5%) had previous illicit treatment. Seven (17.1%) used non-prescribed hormones, and three (7.3%) received silicone breast injections. Two patients presented with symptoms from silicone injections; the remaining were asymptomatic. Six patients underwent subglandular breast augmentation, one had a reduction mammoplasty, and one underwent an implant exchange. Only one patient had postoperative complications that healed without surgical intervention. This series emphasizes the need to assess the prevalence of silicone injections and illicit hormone use among transgender chest feminization patients. The use of these substances was relatively high in our population compared to national averages; however, there is a low risk of postoperative complications. Providers may perform breast surgery in patients with a history of illicit augmentation practices after careful evaluation and surgical planning.

Addressing opioid use disorder among rural pregnant and postpartum women: a study protocol

BackgroundOpioid use disorder (OUD) among women delivering at a hospital has increased 400% from 1999–2014 in the United States. From the years 2007 to 2016, opioid-related mortality during pregnancy increased over 200%, and drug-overdose deaths made up nearly 10% of all pregnancy-associated mortality in 2016 in the US. Disproportionately higher rates of neonatal opioid withdrawal syndrome (NOWS) have been reported in rural areas of the country, suggesting that perinatal OUD is a pressing issue among these communities. There is an urgent need for comprehensive, evidence-based treatment services for pregnant women experiencing OUD. The purpose of this article is to describe a study protocol aimed at developing and evaluating a perinatal OUD curriculum, enhancing evidence-based perinatal OUD treatment in a rural setting, and evaluating the implementation of such collaborative care for perinatal OUD.MethodsThis two-year study employed a one group, repeated measures, hybrid type-1 effectiveness-implementation design. This study delivered interventions at 2 levels, both targeting improvement of care for pregnant women with OUD. The first area of focus was at the community healthcare provider-level, which aimed to evaluate the acceptability and feasibility of perinatal OUD education across time and to improve provider education by increasing knowledge specific to: MOUD provision; screening, brief intervention, and referral to treatment (SBIRT) utilization; and NOWS treatment. The second area of intervention focus was at the patient-level, which assessed the preliminary effect of perinatal OUD provider education in promoting illicit opioid abstinence and treatment engagement among pregnant women with OUD. We adopted constructs from the Consolidated Framework for Implementation Research (CFIR) to assess contextual factors that may influence implementation, and the Reach, Effectiveness, Adoption, Implementation, and Maintenance (RE-AIM) model to comprehensively evaluate implementation outcomes.DiscussionThis article presents the protocol of an implementation study that is employing the CFIR and RE-AIM frameworks to implement and evaluate a perinatal OUD education and service coordination program in two rural counties. This protocol could serve as a model for clinicians and researchers seeking to implement improvements in perinatal care for women with OUD in other rural communities.Trial registration NCT04448015 clinicaltrials.gov.

LC-MS/MS analyses of bile and histological analyses of thymus as diagnostic tools to detect low dose dexamethasone illicit treatment in beef cattle at slaughterhouse

Dexamethasone (DXM) is a synthetic adrenal corticosteroid with anti-inflammatory properties used for therapeutic purposes in a wide range of pathologies and of the most common corticosteroids used for anabolic purposes in beef cattle. It is proven that DXM induces histological changes, traceable as increasing fatty infiltration of the thymus associated with a concurrent decrease of the cortex-medulla ratio, so the histological examination of the thymus gland has been established as an indirect morphological biomarker. The aim of the present study is to compare thymus histology and DXM concentrations in biological fluids collected at slaughterhouse after 1 month of DXM treatment. Our findings demonstrate that a low dosage of DXM administered to 12 months-old-Chianina beef cattle induces severe thymic atrophy with concurrent reduction of the cortex/medulla ratio, demonstrable even when DXM residues are not found in serum and urine samples. It is worth to note that, at the slaughterhouse, DXM residues are detectable in bile samples, indicating the ability of this biological fluid to bio-concentrate the administered drug if compared to serum and urine. Therefore, bile could be candidates as new liquid matrix for the screening programs planned to contrast the illegal use of anabolic substances.

Multi-Analyte MS Based Investigation in Relation to the Illicit Treatment of Fish Products with Hydrogen Peroxide

Fishery products are perishable due to the action of many enzymes, both endogenous and exogenous. The latter are produced by bacteria that may contaminate the products. When fishes age, there is a massive bacteria growth that causes the appearance of off-flavor. In order to obtain “false” freshness of fishery products, an illicit treatment with hydrogen peroxide is reported to be used. Residues of hydrogen peroxide in food may be of toxicology concern. We developed two mass spectrometry based methodologies to identify and quantify molecules related to the treatment of fishes with hydrogen peroxide. With ultra-high-performance liquid chromatography–mass spectrometry (UHPLC-MS) we evaluated the concentration of trimethylamine-N-oxide (TMAO), trimethylamine (TMA), dimethylamine (DMA), and cadaverine (CAD) in fish products. After evaluating LOQ, we measured and validated the lower limits of quantification (LLOQs as first levels of calibration curves) values of 50 (TMAO), 70 (TMA), 45 (DMA), and 40 (CAD) ng/mL. A high ratio between TMAO and TMA species indicated the freshness of the food. With a GC-MS method we confirmed the illicit treatment measuring the levels of H2O2 after an analytical reaction with anisole to give 2-hydroxyanisole as a marker. This latter product was detected in the headspace of the homogenized sample with simplification of the work-up. A LLOQ of 50 ng/mL was checked and validated. When fish products were whitened and refreshed with hydrogen peroxide, the detected amount of the product 2-hydroxyanisole could be very important, (larger than 100 mg/kg). The developed analytical methods were suitable to detect the illicit management of fishery products with hydrogen peroxide; they resulted as sensitive, selective, and robust.

CLASSIFICATION OF EXPERTISES ON THE BASIC SATISFACTION OBJECT OF RESEARCH AND OTHER GROUNDS

The article deals with the new classification of antitort and other expertises on the basis object of the research and other grounds. The classes of criminalistic, medical and narcological expertises and the class of human psychic expertises are distinguished. The essence of the class of criminalistic expertises for the research of any trasessubstances (tracks: imprints, diagnostic and situational displays; substances: objects, devices, mechanisms, substances), relationships or phenomena of importance for overcoming criminal and/or other offenses except, excluding the psyche and metabolic processes of a person, his body, parts or secretions, as well as clothing, footwear and other traction substations in the event of joint causation of bodily harm to a person or other trace formation with his body and/or parts and/or excreta. The list of genuses of criminalistic expertises is given: firearms, ammunition and traces of illicit treatment of them; mechanical weapons, ammunition and traces of illicit handling of them; explosive weapons, explosive devices and substances, means of blasting and traces of the illicit treatment of them; substances, materials and products from them; restoration of destroyed texts and marking symbols; trassological; phonoscopic; commission odorological; handwriting author-identification; portrait; technical; technological, commodity research; accounting; financial-economical, biological, engineering-ecological, art criticism, military, etc., as well as kinds of expertises of some of these genuses. The list of the genuses of medical expertises: genoscopic, immunological, biological, cytological, trassological, toxicological, trace ssubstances, corpses of a person, etc., as well as expertises of the human psyche: a psychological one that should investigate various peculiarities of the human psyche; and psychiatric, who investigates various deviations and diseases of the human psyche, including establishes the fact of the presence and recognition of the need for treatment of various manifestations of psychosociality. The essence of the class of narcological expertises is determined, which must establish the fact of the presence and necessity of treatment of various manifestations of psycho-metabolic dependence on alcoholism, drug addiction, toxicomania, etc. The variants of classification of antitortious and other expert investigations on the sequence of conducting, on the volume of research, on the composition and nature of the special knowledge, on the place of conducting and on the type of procedural proceedings. According to the results of the research, it is suggested to use the above mentioned innovations to develop a solution for this problem, exhaustive in terms of the genuses and types of expertises, in the process of discussion. Keywords: the classification of expert investigations criminalistic, medical and narcological expertises; human psychic expertises.

New surveillance concepts in food safety in meat producing animals: the advantage of high throughput 'omics' technologies - A review.

The misuse of anabolic hormones or illegal drugs is a ubiquitous problem in animal husbandry and in food safety. The ban on growth promotants in food producing animals in the European Union is well controlled. However, application regimens that are difficult to detect persist, including newly designed anabolic drugs and complex hormone cocktails. Therefore identification of molecular endogenous biomarkers which are based on the physiological response after the illicit treatment has become a focus of detection methods. The analysis of the ‘transcriptome’ has been shown to have promise to discover the misuse of anabolic drugs, by indirect detection of their pharmacological action in organs or selected tissues. Various studies have measured gene expression changes after illegal drug or hormone application. So-called transcriptomic biomarkers were quantified at the mRNA and/or microRNA level by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) technology or by more modern ‘omics’ and high throughput technologies including RNA-sequencing (RNA-Seq). With the addition of advanced bioinformatical approaches such as hierarchical clustering analysis or dynamic principal components analysis, a valid ‘biomarker signature’ can be established to discriminate between treated and untreated individuals. It has been shown in numerous animal and cell culture studies, that identification of treated animals is possible via our transcriptional biomarker approach. The high throughput sequencing approach is also capable of discovering new biomarker candidates and, in combination with quantitative RT-qPCR, validation and confirmation of biomarkers has been possible. These results from animal production and food safety studies demonstrate that analysis of the transcriptome has high potential as a new screening method using transcriptional ‘biomarker signatures’ based on the physiological response triggered by illegal substances.

Paraoxonase 1 (PON1) is a valid plasma marker to detect illicit treatment with dexamethasone in veal calves

Paraoxonase 1 (PON1) is a valid plasma marker to detect illicit treatment with dexamethasone in veal calves

Thymus atrophy is an efficient marker of illicit treatment with dexamethasone in veal calves: Results from a triennial experimental study

Glucocorticoids, used in a wide range of pathologies thank to their therapeutical properties, are also illegally used as growth-promoters in animal breeding even if the European Union regulates their use to protect consumers' health from the adverse effects of residues in food.The first aim of the study was to establish the applicability of two histological parameters – atrophy and cortex-medulla ratio - to detect glucocorticoids misuse in calves. The second aim was to concurrently test the potentiality of both parameters to discriminate between treated and untreated animals.One hundred and seventy-two male Friesian veal calves were raised for six months and divided into two groups: Group A (106 calves) was given dexamethasone per os for twenty days (0.4mg/day), Group B (66 calves) used as control. Thymic samples were microscopically examined. Fat infiltration was evaluated and a degree of atrophy, ranging from 1 to 3 (mild, moderate, severe) was attributed; thymic cortex-medulla ratio was calculated too.Fisher's exact test and a Wilcoxon–Mann–Whitney test were performed to investigate the differences in thymic atrophy and cortex-medulla ratio between the groups.Results demonstrate that the thymic atrophy grading was significantly increased in group A (p=0,006), whereas the cortex-medulla ratio was decreased (p<0,004) when compared to group B; moreover, the parallel testing with fixed degree of atrophy and cortex-medulla ratio cut-off thresholds optimize the sensitivity (90%) in the detection of glucocorticoids anabolic treatments.These data suggest that microscopic thymus analysis represent a valid tool for the screening and monitoring of glucocorticoid illicit treatments.

Morphological Examination and Transcriptomic Profiling To Identify Prednisolone Treatment in Beef Cattle.

In livestock production corticosteroids are licensed only for therapy; nevertheless, they are often illegally used as growth promoters. The aim of this study was to identify morphological or biomolecular alterations induced by prednisolone (PDN) in experimentally treated beef cattle, because PDN and its metabolites are no longer detectable by LC-MS/MS methods in biological fluids. Moreover, PDN does not induce any histological alterations in the thymus, different from dexamethasone treatments. Therefore, a marker of illicit treatment for this growth promoter could be useful. Eight male Italian Friesian beef cattle were administered prednisolone acetate 30 mg day-1 per os for 35 days, and seven beef cattle represented the control group. Six days after drug withdrawal, the animals were slaughtered. Morphological and morphometric modifications were evaluated in the epididymis and testis, whereas transcriptomic changes induced by PDN administration were investigated in peripheral blood mononuclear cells (PBMCs) at different sampling times and in skeletal muscle and testis sampled at slaughtering. In the epididymis, spermatozoa number decreased in PDN-treated animals, and in some cases they were totally absent. Correspondingly, in the testis of treated animals, down-regulation for serine/threonine kinase 11 (STK11) gene expression was detected (p < 0.01). DNA microarray analysis revealed a total of 133 differentially expressed genes in skeletal muscle and testis, and 907 and 1416 in PBMCs after 33 days of treatment and at slaughtering, respectively. Histological investigations on epididymal content could represent a promising marker for PDN treatment in beef cattle and could be used as a screening method to identify animals worthy of further investigation with official methods. Moreover, the clear transcriptomic signature of PDN treatment evidenced in PBMCs supported the possibility of using this matrix to monitor the illicit treatment in vivo during ranching.

A Liquid Chromatography-Tandem Mass Spectrometry Method for the Detection of Antimicrobial Agents from Seven Classes in Calf Milk Replacers: Validation and Application.

Calf milk replacers are low-cost feeds that contain available, digestible protein. During their reconstitution, however, the addition of drugs, such as antibiotics, could make them a very simple route for illicit treatment for therapeutic, preventive, or growth-promoting purposes. We developed an HPLC-MS/MS method, preceded by a unique extraction step, able to identify 17 antibiotics from seven classes (penicillins, tetracyclines, fluoroquinolones, sulfonamides, cephalosporins, amphenicols, and lincosamides) in this matrix. Prior to solid phase extraction (SPE), the sample underwent deproteinization and defatting. The method was fully validated according to Commission Decision 2002/657/EC. Decision limits (CCα) and detection capability (CCβ) were in the ranges of 0.13-1.26 and 0.15-1.47 ng/mL, respectively. Thirty-eight samples were finally analyzed, showing the occasional presence of marbofloxacin (six samples) and amoxicillin (one sample).

Quantification of natural and synthetic glucocorticoids in calf urine following different growth-promoting prednisolone treatments

Over the last few years, low levels of prednisolone have been reported in several cattle urine samples by a number of laboratories within the EU at an average concentration of 2.0ngmL−1. The occurrence of prednisolone residues together with increased levels of hydrocortisone and cortisone in urine and tissue samples of untreated animals seems to demonstrate that traces of this steroid can be produced endogenously during stressful situations. Therefore, the endogenous origin of prednisolone makes difficult to correlate positive samples to a potential illicit treatment. An experimental study was developed to investigate the presence of natural and synthetic glucocorticoids and to evaluate levels of excreted prednisolone following growth-promoting treatments. Urine samples from calves undergone oral treatment with prednisolone, alone and in association with dexamethasone, were analyzed by a LC–MS/MS method, validated according to the Commission Decision 2002/657/EC. We also investigated if urinary free 6β-hydroxyhydrocortisone/hydrocortisone ratio could be a reliable biomarker of illicit treatment with prednisolone and dexamethasone in calves. Our data revealed that urinary levels of prednisolone after both oral prednisolone treatments, never exceeded the value of 1.1ngmL−1. Similar prednisolone levels were found in urine samples of untreated calves. Moreover the presence of 6β-hydroxyhydrocortisone below the CCα value made possible to estimate the 6β-hydroxyhydrocortisone/hydrocortisone ratio only in a very limited number of samples. Obtained data suggest that further criteria have to be considered to allow correct decisions about the urinary presence of prednisolone during control activities.

Pseudoendogenous presence of β-boldenone sulphate and glucuronide in untreated young bulls from the food chain

The administration of boldenone (bold) to bovines, either for growth promotion or therapeutic purposes, has been banned in the EU since 1981. It is, however, a pseudoendogenous hormone, thus its detection in bovine urine, in the form of α-boldenone conjugates, is considered fully compliant up to 2 ng ml−1. Greater attention has been placed on β-boldenone, the anabolic active epimer, whose conjugated form must be absent in urine. Recently, the identification of a biomarker representing unquestionable evidence of illicit treatment with bold or its precursor androstadienedione has been a major topic in the literature regarding the detection of residues in bovine urine, and β-boldenone sulphate is a candidate molecule. In this study, we used a method previously validated according to the European Commission Decision 2002/657/EC for the determination of sulphate and glucuronide conjugates of β-boldenone. We assessed the occurrence of these molecules in young bull urine, with the aim of understanding whether they could be of endogenous origin, and to check for a possible relationship with particular environmental and stress conditions. Urine samples from 56 young bulls were collected after transport stress, under non-stressful conditions and after transport and slaughter stress. Histopathological investigation of the hormone target organs, i.e. the bulbourethral and prostate glands, was also performed. The results indicate an inverse relationship between the presence and concentration of β-boldenone sulpho- and gluco-conjugates in urine, and stress conditions, expressed by the absence of detection at the slaughterhouse. No significant macroscopic and histologic lesions were detected. Our study indicates that β-boldenone sulphate could be a biomarker of treatment only at the slaughterhouse, while at the farm, in untreated animals (i.e. after a five-month period under the control of Official Veterinarians), sulphate and glucuronide metabolites were found with a frequency of 78% and 46%, respectively, showing the endogenous origin of boldenone.