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Background: Vulvar squamous cell carcinoma (VSCC) has been recently associated with Equus caballus papillomavirus type 2 (EcPV2) infection. Still, few reports concerning this disease are present in the literature. Objective: To describe a case of naturally occurring EcPV2‐induced VSCC, by investigating tumour ability in undergoing the epithelial‐to‐mesenchymal transition (EMT). Study design: Case report. Methods: A 13‐year‐old Haflinger mare was referred for a rapidly growing vulvar mass. After surgical excision, the mass was submitted to histopathology and molecular analysis. Histopathological diagnosis was consistent with a VSCC. Real‐time qPCR, real‐time reverse transcriptase (RT)‐qPCR and RNAscope were carried out to detect EcPV2 infection and to evaluate E6/E7 oncogenes expression. To highlight the EMT, immunohistochemistry (IHC) was performed. Expression of EMT‐related and innate immunity‐related genes was investigated through RT‐qPCR. Results: Real‐time qPCR, RT‐qPCR and RNAscope confirmed EcPV2 DNA presence and expression of EcPV2 oncoproteins (E6 and E7) within the neoplastic vulvar lesion. IHC highlighted a cadherin switch together with the expression of the EMT‐related transcription factor HIF1α. With RT‐qPCR, significantly increased gene expression of EBI3 (45.0 ± 1.62, p < 0.01), CDH2 (2445.3 ± 0.39, p < 0.001), CXCL8 (288.7 ± 0.40, p < 0.001) and decreased gene expression of CDH1 (0.3 ± 0.57, p < 0.05), IL12A (0.04 ± 1.06, p < 0.01) and IL17 (0.2 ± 0.64, p < 0.05) were detected. Main limitations: Lack of ability to generalise and danger of over‐interpretation. Conclusion: The results obtained were suggestive of an EMT event occurring within the neoplastic lesion. [ABSTRACT FROM AUTHOR]

Simple Summary: Reptiles usually carry Salmonella without showing any signs of infection. In this case, an adult male corn snake died 48 h after eating a feeder mouse bought online. The snake and mouse livers tested positive for Salmonella, specifically Salmonella enterica subsp. enterica serovar Midway. A genome analysis revealed that the two samples were from the same bacterial strain, and both had genes responsible for the bacteria's virulence. This report is about a pet snake that acquired Salmonella from a feeder mouse and then died from septic shock. It shows how feeder mice can be a source of clinical salmonellosis in pet snakes, which can pose a risk to humans too. Reptiles are usually asymptomatic carriers of Salmonella, with the manifestation of typical clinical signs of acute forms in adult and non-immunocompromised animals being considered exceptions. In the present case, an adult male corn snake (Pantherophis guttatus) was found dead due to septic shock 48 h after consuming a feeder mouse purchased online. The snake's tissue samples and faeces were cultured for bacteria isolation. Microbiological examinations of the snake and mouse livers revealed the presence of Salmonella enterica subsp. enterica serovar Midway. A whole-genome analysis of these two isolates showed a high correlation between them: they belonged to the strain type ST-357 for the classic MLST scheme and to the strain type ST 171322 for the cgMLST scheme. Also, a virulence gene analysis revealed the presence of stdB and STM3026 genes. This report conveys a case of food-borne salmonellosis in a pet snake, transmitted from a feeder mouse, likely responsible for the snake's death due to septic shock. It highlights the relevance of feeder mice as a source of Salmonella infections in snakes and the associated risks to human health. [ABSTRACT FROM AUTHOR]

Vulvo-vaginal epithelial tumors are uncommon in mares, and data on the epithelial-to-mesenchymal transition (EMT) and the tumor-immune microenvironment (TIME) are still lacking. This is a study investigating the equus caballus papillomavirus type 2 (EcPV2) infection state as well as the EMT process and the tumor microenvironment in vulvo-vaginal preneoplastic/ benign (8/22) or malignant (14/22) epithelial lesions in mares. To do this, histopathological, immunohistochemical, transcriptomic, in situ hybridization, and correlation analyses were carried out. Immunohistochemistry quantification showed that cytoplasmic E-cadherin and β-catenin expression as well as nuclear β-catenin expression were features of malignant lesions, while benign/preneoplastic lesions were mainly characterized by membranous E-cadherin and β-catenin expression. Despite this, there were no differences between benign and malignant equine vulvo-vaginal lesions in the expression of downstream genes involved in the canonical and noncanonical wnt/β-catenin pathways. In addition, malignant lesions were characterized by a lower number of cells with cytoplasmic cytokeratin expression as well as a slightly higher cytoplasmic vimentin immunolabeling. The TIME of malignant lesions was characterized by more numerous CD204+ M2-polarized macrophages. Altogether, our results support the hypothesis that some actors in TIME such as CD204+ M2-polarized macrophages may favor the EMT process in equine vulvo-vaginal malignant lesions providing new insights for future investigations in the field of equine EcPV2-induced genital neoplastic lesions. [ABSTRACT FROM AUTHOR]

Simple Summary: Testosterone and nandrolone can be illegally administered to meat-producing animals as synthetic esters. To tackle the abuse of growth promoters, alternative approaches able to investigate specific changes induced in proteins, transcripts, and metabolites are becoming recommendable. This work aimed to characterize transcriptome perturbations related to the illicit administration of steroid esters in fattening pigs. Animals were treated with testosterone esters (Sustanon®, Organon, Jersey City, NJ, USA) or nandrolone esters (Myodine®, Le Vet Beheer B.V., Oudewater, Utrecht, The Netherlands). At the end of the trial, liver samples were collected for gene expression studies. Comparisons between treated and control groups using RNAseq allowed the identification of 491 differentially expressed genes (DEGs). Further analysis of DEGs characterized a smaller cluster of 16 genes. A field survey performed on liver samples collected from pigs belonging to different breeds and weight categories allowed the validation of the selected biomarkers using qPCR, confirming their specificity by comparison with testosterone residue profiles on respective serum samples. The use of anabolic–androgenic steroids (AASs) as growth promoters in farm animals is banned in the European Union, representing both an illicit practice and a risk for consumer health. However, these compounds are still illegally administered, often in the form of synthetic esters. This work aimed to characterize significant coding RNA perturbations related to the illicit administration of testosterone and nandrolone esters in fattening pigs. A total of 27 clinically healthy 90-day-old pigs were randomly assigned to test and control groups. Nine animals were treated with testosterone esters (Sustanon®) and other nine with nandrolone esters (Myodine®). At the end of the trial, liver samples were collected and analyzed using RNAseq, allowing the identification of 491 differentially expressed genes (DEGs). The transcriptional signature was further characterized by a smaller sub-cluster of 143 DEGs, from which a selection of 16 genes was made. The qPCR analysis confirmed that the identified cluster could still give good discrimination between untreated gilt and barrows compared to the relative testosterone-treated counterparts. A conclusive field survey on 67 liver samples collected from pigs of different breeds and weight categories confirmed, in agreement with testosterone residue profiles, the specificity of selected transcriptional biomarkers, showing their potential applications for screening purposes when AAS treatment is suspected, allowing to focus further investigations of competent authorities and confirmatory analysis where needed. [ABSTRACT FROM AUTHOR]

Among forbidden substances included in the European Union legislation, endogenous steroids constitute a challenge in the framework of veterinary Official Monitoring Plans. They can be naturally present in body fluids at variable levels depending on the species, sex and age of the animals. Considering the significant advances achieved in breeding conditions and in the selection of producing traits in meat cattle, the aim of this study was to verify by analytical method, in veal calves housed under controlled conditions, if the level of natural steroids hormones assumed as physiological are still actual. The second aim of the study was to verify if the normal histological pattern of growth promoters in target organs is influenced accordingly. Bovine male sex organs are currently analysed in the frame of the Italian histological plan to monitor illicit treatments trend, highlighting microscopic, induced alterations. The levels of 17β-estradiol and progesterone residues resulted under the Limit of Quantitation of the approved official methods and the level of testosterone resulted below the level stated in the Italian Ministerial Decree in force. Male target organs appeared within the limits of the standard histological features. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Plastic is a polymer extremely resistant to degradation that can remain for up to hundreds or thousands of years, leading to the accumulation of massive amounts of plastic waste throughout the planet's ecosystems. Due to exposure to various environmental factors, plastic breaks down into smaller particles named microplastics (1–5000 μm) and nanoplastics (<1 μm). Microplastics (MPs) are ubiquitous pollutants but, still, little is known about their effects on human and animal health. Herein, our aim is to investigate cytotoxicity, oxidative stress, inflammation and correlated gene modulation following exposure to polystyrene microplastics (PS-MPs) in HRT-18 and CMT-93 epithelial cell lines. After 6, 24 and 48 h PS-MPs treatment, cell viability (MTT) and oxidative stress (SOD) assays were performed; subsequently, expression changes and cytokines release were investigated by Real-Time PCR and Magnetic-beads panel Multiplex Assay, respectively. For each exposure time, a significantly increased cytotoxicity was observed in both cell lines, whereas SOD activity increased only in CMT-93 cells. Furthermore, Magnetic-beads Multiplex Assay revealed an increased release of IL-8 in HRT-18 cells' medium, also confirmed by gene expression analysis. Results obtained suggest the presence of a pro-inflammatory pattern induced by PS-MPs treatment that could be related to the observed increase in cytotoxicity. [ABSTRACT FROM AUTHOR]

This article discusses the challenges of detecting food fraud and ensuring the authenticity and traceability of food products. It highlights that food products are complex and can come from different geographical areas, which can affect their composition. The globalization of supply chains and weak governance contribute to the incidence of food irregularities. The article explores various spectroscopic and chromatographic techniques used to study the authentication and traceability of food, such as near-infrared, mid-infrared, Raman, absorption ultraviolet-visible, nuclear magnetic resonance, and mass spectrometry. The development of innovative methods to test the authenticity and traceability of foods is crucial in preventing food fraud and protecting consumers and food producers. [Extracted from the article]

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. [ABSTRACT FROM AUTHOR]

Fish species substitution is one of the most common forms of fraud all over the world, as fish identification can be very challenging for both consumers and experienced inspectors in the case of fish sold as fillets. The difficulties in distinguishing among different species may generate a "grey area" in which mislabelling can occur. Thus, the development of fast and reliable tools able to detect such frauds in the field is of crucial importance. In this study, we focused on the distinction between two flatfish species largely available on the market, namely the Guinean sole (Synaptura cadenati) and European plaice (Pleuronectes platessa), which are very similar looking. Fifty fillets of each species were analysed using three near-infrared (NIR) instruments: the handheld SCiO (Consumer Physics), the portable MicroNIR (VIAVI), and the benchtop MPA (Bruker). PLS-DA classification models were built using the spectral datasets, and all three instruments provided very good results, showing high accuracy: 94.1% for the SCiO and MicroNIR portable instruments, and 90.1% for the MPA benchtop spectrometer. The good classification results of the approach combining NIR spectroscopy, and simple chemometric classification methods suggest great applicability directly in the context of real-world marketplaces, as well as in official control plans. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

With the aim of specifically investigating patterns associated with three steroid treatments (17β‐nandrolone, 17β‐estradiol, and 17β‐nandrolone + 17β‐estradiol) in bovine, an reversed phase liquid chromatography (RPLC)‐electrospray ionization (ESI)(+/−)‐high‐resolution mass spectrometry (HRMS) study was conducted to characterize the urinary profiles of involved animals. Although specific fingerprints with strong differences could be highlighted between urinary metabolite profiles within urine samples collected on control and treated animals, it appeared further that significant discriminations could also be observed between steroid treatments, evidencing thus specific patterns and candidate biomarkers associated to each treatment. An MS‐2 structural elucidation step enabled level‐1 identification of two biomarkers mainly involved in energy pathways, in relation to skeletal muscle functioning. These results make it possible to envisage a global strategy for the detection of anabolic practices involving steroids, while at the same time providing clues as to the compounds used, which would facilitate the confirmation stage to follow. [ABSTRACT FROM AUTHOR]

Abstract Background Routine rapid testing for Bovine Spongiform Encephalopathy (BSE) has highlighted some problems with BSE rapid test performance, the most significant being the number of initially reactive samples and the false positive results on autolyzed tissue. This point is important for BSE active surveillance in risk populations, because tissue autolysis is often unavoidable in routine cases. A robust test suitable for use on field material is therefore needed. To date, very limited information regarding the effect of autolysis on the robustness of rapid tests has been documented; therefore, the National Reference Centre for Animal Encephalopathies (CEA) rapid test laboratory selected 450 autolyzed and negative brain stem samples from fallen stock bovines older than 24 months to assess the specificity of four tests approved for BSE active surveillance: Biorad TeSeE, Enfer TSE version 2.0, Prionics® Check LIA, and IDEXX Herd Check BSE Antigen Kit EIA. The samples were graded according to the degree of autolysis and then dissected into five portions, four of which randomly assigned to processing by rapid tests and one to be available for confirmatory Western blot analysis. Findings The specificity of the four systems was 100% for all three grades of autolysis, while the percentage of initially reactive results was 0.00 (95%CI 0.00-0.82), 0.22 (95%CI 0.006-1.23), 0.44 (95%CI 0.05-1.60), and 0.89 (95%CI 0.24-2.26) for the Biorad TeSeE, the Prionics® Check LIA, the IDEXX Herd Check BSE and the Enfer TSE tests, respectively. No association with the degree of autolysis could be drawn. Conclusions The present study demonstrates that the four rapid tests can be considered well-running diagnostic tools regardless of tissue quality; nevertheless, the number of initial reactive samples reported for some systems must not be underestimated in routine testing. Furthermore the compliance with the reported performance can be guaranteed only when an ongoing high careful batch quality control system is in place.

Background. Sex steroids administration in meat producing animals is forbidden within the EU to preserve consumers' safety, but continuous monitoring to identify resurgence of their misuse is needed. Among biomarkers related to sex steroids abuse in veal calves the regucalcin (RGN)mRNAperturbations in testis have been described in RNAlater samples. To setup novel diagnostic method, to update current tests available in National Residue Control Plans (NRCPs) and in legal dispute when illicit practices on farm animals are suspected, the reliability of RGN profiling was assessed by histological and molecular techniques. Methods. Formalin fixed paraffin embedded (FFPE) testis samples, chosen being the most effective preservation strategy adopted by histological NRCPs and allowing easier retrospective analysis if required by legal disputes, were analyzed from veal calves treated with nandrolone, 17β-estradiol and a cocktail of the two hormones. RGN levels were determined by quantitative Real Time PCR and Immunohistochemistry assays. Test performances were assessed and compared by multiple ROC curves. Results. Both tests resulted sensitive and specific, allowing to enrich, in future field investigation, novel integrated diagnostic protocols needed to unveil sex steroid abuse. Discussion. Developed RT-qPCR and IHC methods confirmed RGN as a useful and robust biomarker to detect illegal administration of sex steroid hormones in veal calves. The developed methods, successfully applied to ten years old FFPE blocks, could allow both retrospective analysis, when supplementary investigations are requested by authorities, and future implementation of current NRCPs. [ABSTRACT FROM AUTHOR]

The issue of food safety has acquired increased importance, and fraud is a major concern for the food industry. Among different types of food adulteration, there is the sale of frozen-thawed smoked salmon product as fresh, which not only decreases the quality of products but also misleads consumers and may involve associated health risks. In response to this problem, we tested the performance of histology to identify smoked salmon as fresh or frozen-thawed as a valid analytical method, so food business operators and official controllers can reliably and correctly classify the storage state of the product. Three groups of samples were prepared: group A (n = 36), fresh samples; group B (n = 36), frozen at −18°C for 30 days; and group C (n = 36), stored at −3°C for 30 days after packaging. Two histopathologists examined all samples in blind evaluations and classified them as fresh or frozen-thawed. Sensitivity, specificity, and interrater agreement were calculated. Results show high performance with the test: 80.6% sensitivity (95% confidence intervals [CI]: 64 to 91.8%); 95.6% specificity (95% CI: 89.1 to 98.8%); and Cohen's kappa was 0.81 (95% CI: 0.64 to 0.98%). Histology is a reliable and highly accurate method to differentiate fresh from frozen-thawed smoked salmon and could be used by the industry and official controllers to verify the labeling of the commercial product. [ABSTRACT FROM AUTHOR]

Member States of the EU are required to monitor the use of pharmacologically active substances in food-producing animals. There is evidence, however, that the target-based approach currently applied in official monitoring plans might under-estimate the real incidence of growth promoter abuse in livestock. As demonstrated for sex hormones, the association of effect-oriented biological screening with chemical confirmatory techniques could be the best strategy in revealing the abuse of veterinary drugs. Here we demonstrate the reliability of a cell-based assay to screen calf urine samples for synthetic glucocorticoids. The validation included the most widely used synthetic drugs (flumethasone, dexamethasone, betamethasone, methylprednisolone and prednisolone) and was developed according to the Commission Decision 2002/657/EC, thus including the verification of cut-off level, the β error, the specificity, ruggedness and stability. The study was carried out using prednisolone as representative substance at 5 ng mL−1 concentration. All blank and spiked urine fulfilled the EU criteria, moreover the method resulted in being specific and sound, and the analytes in urine were stable for at least 30 days. The assay results indicated its suitability for a qualitative analysis of calf urine samples. This method enabled the detection of low doses of synthetic glucocorticoids (GCs) in matrix (<2 ng mL−1 for flumethasone, dexamethasone, betamethasone; < 4 ng mL−1 for methylprednisolone; 5 ng mL−1 for prednisolone), with the possibility of detecting new or unknown molecules and cumulative effects of low-level mixtures with glucocorticoid bioactivity. [ABSTRACT FROM AUTHOR]

The illegal use of glucocorticoids (GCs) as growth-promoters (GPs) is prohibited in farm animals in the European Union because the strong pharmacological activity of most synthetic GCs produces residues that are dangerous for human consumption. Among the alternative methods proposed to increase the efficacy of official controls, histology was the technique of choice in Italy on account of its high performance level. The aim of this study was to evaluate the reliability of immunohistochemistry (IHC) using anti-cleaved-Lamin A antibody to enhance the performance of the histological test applied to GC-related microscopic changes in the thymus. Veal calves (VC) and beef cattle (BC) were raised and both underwent different growth-promoting protocols or were left untreated. The morphology of the thymus parenchyma was evaluated for cortical atrophy with concurrent adipose tissue infiltration, and a score of 1 to 3 was attributed. A semi-quantitative IHC analysis was also performed by counting the number of positive thymocytes in 5 randomly selected high-power fields (HPFs). The distribution of the thymus atrophy scores was significantly different among the subgroups in both BC and VC. The IHC values were higher in untreated than in treated animals, for both BC and VC. The association between thymus atrophy score and IHC positivity showed higher median values in control than in treated animals (independently of the treatment protocol), for both BC and VC. Our data shows that IHC against anti-cleaved-Lamin A antibody is a reliable marker to detect illegal GC treatments, administered either alone or in association with other growth promoters, in both BC and VC. Combining IHC with the thymus atrophy score improves the accuracy of the histological method in correctly identifying treated animals and could represent a valuable, reproducible method to be applied to large-scale screening programmes. [ABSTRACT FROM AUTHOR]

Within the particular context of controlling chemical residues in food, an alternative to targeted approaches has emerged; it consists in the characterisation of physiological perturbations induced upon exposure of animals to a given chemical substance/class of substances to highlight suitable biomarkers addressing safety and/or regulatory issues. Metabolomics in particular has been investigated in the hope of identifying such biomarkers, and a range of studies have demonstrated the efficiency of the strategy. Until very recently, steps remained to be taken towards official or commercial implementation of corresponding tools. In particular, the lack of guidelines and criteria to validate such methods that do not target specific chemical species per se, constituted a bottleneck. In the present work, a metabolomics model dedicated to the detection of β-agonist administration in bovines has been developed and fully validated; criteria (selectivity, robustness, stability, suspicion threshold definition, false positive and false negative rates) have been proposed in agreement with EU expectations (Dec 2002/657), enabling demonstration that performances comply with screening requirements. Although some of the biomarkers involved in the prediction model remain un-elucidated, the corresponding LC-HRMS method has recently been ISO17025 accredited, allowing for the very first official implementation of a metabolomics based strategy within French National Monitoring Plans. [ABSTRACT FROM AUTHOR]

Background: Canine cancer registry data can be put to good use in epidemiological studies. Quantitative comparison of tumour types may reveal unusual cancer frequencies, providing directions for research and generation of hypotheses of cancer causation in a specific area, and suggest leads for identifying risk factors. Here we report canine cancer incidence rates calculated from a population-based registry in an area without any known specific environmental hazard. Results: In its 90 months of operation from 2001 to 2008 (the observation period in this study), the populationbased Piedmont Canine Cancer Registry collected data on 1175 tumours confirmed by histopathological diagnosis. The incidence rate was 804 per 100,000 dog-years for malignant tumours and 897 per 100,000 dog-years for benign tumours. Higher rates for all cancers were observed in purebred dogs, particularly in Yorkshire terrier and Boxer. The most prevalent malignant neoplasms were cutaneous mastocytoma and hemangiopericytoma, and mammary gland complex carcinoma and simplex carcinoma. Conclusions: The Piedmont canine cancer registry is one of few of its kind whose operations have been consistently supported by long-term public funding. The registry-based cancer incidence rates were estimated with particular attention to the validity of data collection, thus minimizing the potential for bias. The findings on cancer incidence rates may provide a reliable reference for comparison studies. Researches conducted on dogs, used as sentinels for community exposure to environmental carcinogens, can be useful to detect excess risks in the incidence of malignant tumours in the human population. [ABSTRACT FROM AUTHOR]

Under current European Union legislation the use of anabolic steroids in food-producing livestock is banned because of their long-term adverse effects on human health. We examined the expression profile of the immunohistochemical marker progesterone receptor in veal calves’ sex accessory glands following experimental administration of anabolic compounds. The aim was to confirm the accuracy of the immunohistochemical approach in the detection of the over-expression of the progesterone receptor induced by the administration of sexual steroids at low levels (17β-estradiol and nandrolone alone or in combination). A total of 217 male veal calves were randomly divided into four groups: group A (104 calves) treated with 17β-estradiol (5 mg/head; 4 weekly injections); group B (20 calves) treated with nandrolone (50 mg/head; 4 weekly injections); group C (20 calves) treated with the association of the two steroids (5 mg estradiol + 50 mg nandrolone; 4 weekly injections); and group K (73 calves) kept as a control. All the sexual accessory glands were collected at the slaughterhouse (15 days after the last administration) and subjected to immunohistochemical staining with anti-progesterone receptor antibody. All the calves treated with 17β-estradiol alone or in association with nandrolone (groups A and C) showed strong positivity, while nandrolone-treated calves and controls (groups B and K) gave negative results to the immunohistochemical investigation. The statistical analysis showed that the progesterone receptor is a significant predictor of 17β-estradiol treatment alone or in association with nandrolone (p < 0.001): the immunohistochemical study resulted in 100% sensitivity (CI = 95%: 97.1–100%) and specificity (CI = 95%: 95.1–100%) for prostate and 99% sensitivity (CI = 95%: 95.6–100%) and 100% specificity (CI = 95%: 95.1–100%) for bulbo-urethral glands. The data confirm that this innovative biological approach offers a reliable tool to enhance the efficacy of the histological test to detect illegal treatments with estrogens alone or in association with androgens. [ABSTRACT FROM AUTHOR]

Challenges to testing for the illicit use of anabolic substances in meat-producing animals stem from the production of new synthetic compounds and the administration of low-dose cocktails to circumvent detection by the surveillance schemes of European Union member states. This work evaluated for the first time GR-CALUX, a highly sensitive reporter gene assay, as a screening tool for the detection of synthetic glucocorticoids in bovine urine. In order to verify the effect of natural corticosteroids on the method, the bioassay was tested first using blank urine samples collected at the farm and the slaughterhouse. Next, the dose–response curves were measured for the most commonly used synthetic glucocorticoids. The bioassay’s ability to detect them in spiked and incurred samples of bovine urine was then evaluated. Finally, its performance was compared against a commercially available ELISA kit ordinarily used in screening activities. GR-CALUX performance did not appear to be influenced by physiological levels of endogenous corticosteroids in the farm samples, whereas an increase in these hormones might invalidate the analysis in samples obtained at the slaughterhouse. Using pure compounds, GR-CALUX showed a high sensitivity toward the synthetic glucocorticosteroids tested in order of relative potencies: flumethasone ≫ dexamethasone > betamethasone > methylprednisolone > prednisolone. As expected, the bioassay failed to detect the prohormone prednisone. The results obtained from analysis of the spiked and incurred specimens reproduced those of the blank samples and the pure compounds. GR-CALUX is a promising screening tool for the detection of illicit treatments in meat-producing bovines. Its ability to detect the most commonly used synthetic glucocorticoids was comparable with the ELISA test. Importantly, it appeared to be less susceptible to matrix effects than ELISA. [ABSTRACT FROM AUTHOR]

Recombinant bovine growth hormone (rbGH) is produced in large quantities and widely used in a number of countries worldwide to stimulate milk production in dairy animals. The use of this compound in animal production is strictly regulated by food safety directives in force, in particular in the European Union (EU). Although analytical strategies for the detection of rbGH in blood have been successfully reported over the past 15 years, they do not fully answer the expectations of either competent authorities or industrials that would expect measuring its occurrence directly in the milk. As a matrix of excretion but also of consumption, milk appears indeed as the matrix of choice for detecting the use of rbGH in dairy animals. It also allows large volumes to be collected without presenting an invasive character for the animal. However, rbGH detection in milk presents several challenges, mainly related to the sensitivity required for its detection in a complex biological matrix. This review article presents the specific difficulties associated with milk and provides an overview of the analytical strategies reported in the literature and whether they concern indirect or direct approaches to the detection of rbGH administration to animals, with applications either for screening or confirmation purposes. [ABSTRACT FROM AUTHOR]

Background: The illegal administration of hormones, steroids, β-agonists and other anabolic agents to productive livestock in the European Union continues, despite the long-term ban on their use and despite the measures provided under the directives to monitor certain substances and residues thereof in the interest of protecting consumer health and animal wellbeing. Often administered in low doses in the form of a drug cocktail, these compounds escape detection by common analytical techniques. The aim of this study was to determine whether low-dose dexamethasone administration (0.4 mg orally per day, for 20 days) in white-meat calves produced variations in blood coagulation, as measured by thromboelastometry. A second aim was to determine whether such variations could be valid in detecting illicit low-dose dexamethasone treatment. Results: The study population was 42 Friesian calves kept under controlled conditions until 6 months of age. The calves were subdivided into 2 groups: a control group (group A, n = 28) and a group treated with dexamethasone (group B, n = 14) for 20 days beginning at 5 months of age. When compared against the age-matched control group, the dexamethasone-treated calves showed a significant increase in alpha angle, maximum clot firmness and a significant decrease in clot formation time on all thromboelastometric profiles (P < 0.05). The clotting time was significantly decreased on the in-TEM® profile but increased on the ex-TEM® and fib-TEM® profiles (P <0.05). The Receiver Operating Characteristic curves, plotted for the Maximum Clot Elasticity (MCE), had a cut-off value =488.23 mm for in-TEM® MCE [Se 85.7%, (95% CI 57.2-98.2); Sp 100% 96.43% (95% CI 81.7-99.9] and a cut-off value =63.94 mm for fib-TEM MCE® [Se 92.8 (95% CI 66.1-99.8); Sp 89.3% (95% CI 71.8-97.7)]. In order to increase the sensitivity of the test two parameters (in-TEM® and fib-TEM® MCE) were used as two parallel tests; subsequently, the sensitivity rose to a point value of 99% (95% CI 85.4-99.9). Conclusions: Thromboelastometry identified a state of hypercoagulability in the dexamethasone-treated subjects. Furthemore, the results of this preliminary study suggest that TEM may be useful in the detection of illicit low-dose dexamethasone treatment. [ABSTRACT FROM AUTHOR]

Background: Routine rapid testing for Bovine Spongiform Encephalopathy (BSE) has highlighted some problems with BSE rapid test performance, the most significant being the number of initially reactive samples and the false positive results on autolyzed tissue. This point is important for BSE active surveillance in risk populations, because tissue autolysis is often unavoidable in routine cases. A robust test suitable for use on field material is therefore needed. To date, very limited information regarding the effect of autolysis on the robustness of rapid tests has been documented; therefore, the National Reference Centre for Animal Encephalopathies (CEA) rapid test laboratory selected 450 autolyzed and negative brain stem samples from fallen stock bovines older than 24 months to assess the specificity of four tests approved for BSE active surveillance: Biorad TeSeE, Enfer TSE version 2.0, Prionics® Check LIA, and IDEXX Herd Check BSE Antigen Kit EIA. The samples were graded according to the degree of autolysis and then dissected into five portions, four of which randomly assigned to processing by rapid tests and one to be available for confirmatory Western blot analysis. Findings: The specificity of the four systems was 100% for all three grades of autolysis, while the percentage of initially reactive results was 0.00 (95%CI 0.00-0.82), 0.22 (95%CI 0.006-1.23), 0.44 (95%CI 0.05-1.60), and 0.89 (95%CI 0.24-2.26) for the Biorad TeSeE, the Prionics® Check LIA, the IDEXX Herd Check BSE and the Enfer TSE tests, respectively. No association with the degree of autolysis could be drawn. Conclusions: The present study demonstrates that the four rapid tests can be considered well-running diagnostic tools regardless of tissue quality; nevertheless, the number of initial reactive samples reported for some systems must not be underestimated in routine testing. Furthermore the compliance with the reported performance can be guaranteed only when an ongoing high careful batch quality control system is in place. [ABSTRACT FROM AUTHOR]

European Commission Regulation (EU) 1276/2011 requires that fishery products intended for raw consumption be frozen at --20°C for not less than 24 h or at --35°C for at least 15 h in order to kill viable parasites other than trematodes. But because marinating processes are not always effective in destroying nematode larvae, raw marinated fish preparations should be frozen before consumption. This study evaluated the performance of a standardized histological method to distinguish between fresh and frozen-thawed raw marinated fish. Sixty anchovy (Engraulis encrasicolus) fillets were sampled: 30 were marinated at -t-4°C for 24 h, and 30 were frozen at --20°C for 24 h before being marinated for 24 h. All 60 samples were fixed in formalin, processed for paraffin embedding, cut, and stained with hematoxylin and eosin. The slide preparations were examined microscopically by three independent histopathologists and classified as frozen-thawed or negative according to standard operating procedure criteria in use at our laboratory. Performance evaluation of the method showed 100% sensitivity (95% confidence interval [Cl], 88.4 to 100%) and 100% specificity (95% Cl, 88.4 to 100%), and the interrater agreement (Cohen's kappa) was 1 (95% Cl, 0.85 to 1). Histology proved a valid and reliable tool to distinguish fresh from frozen-thawed marinated fish. It can be applied to deliver safe raw fishery products to consumers in order to minimize the risk of anisakidosis. [ABSTRACT FROM AUTHOR]

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, β 2 -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 B 2 -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. [Display omitted] • Novel indirect detection of growth promoters residues in food is needed. • Transcriptional biomarkers profiling has reached a good grade of standardization. • A qPCR array was developed to work on FFPE samples collected in Italian NRCP. • Developed PLS-DA models allowed to setup alternative screening strategies. [ABSTRACT FROM AUTHOR]

The sale of frozen–thawed fish and fish products, labeled as fresh, is currently one of the most common and insidious commercial food frauds. For this reason, the demand of reliable tools to identify the storage conditions is increasing. The present study was performed on two species, commonly sold in large-scale distribution: Cuttlefish (Sepia officinalis) and musky octopus (Eledone spp.). Fifty fresh cephalopod specimens were analyzed at refrigeration temperature (2 ± 2 °C), then frozen at −20 °C for 10 days and finally thawed and analyzed again. The performance of three near-infrared (NIR) instruments in identifying storage conditions were compared: The benchtop NIR Multi Purpose Analyzer (MPA) by Bruker, the portable MicroNIR by VIAVI and the handheld NIR SCiO by Consumer Physics. All collected spectra were processed and analyzed with chemometric methods. The SCiO data were also analyzed using the analytical tools available in the online application provided by the manufacturer to evaluate its performance. NIR spectroscopy, coupled with chemometrics, allowed discriminating between fresh and thawed samples with high accuracy: Cuttlefish between 82.3–94.1%, musky octopus between 91.2–97.1%, global model between 86.8–95.6%. Results show how food frauds could be detected directly in the marketplace, through small, ultra-fast and simplified handheld devices, whereas official control laboratories could use benchtop analytical instruments, coupled with chemometric approaches, to develop accurate and validated methods, suitable for regulatory purposes. [ABSTRACT FROM AUTHOR]

• An available assay to detect sex-steroids abuse in calves was modified to work on FFPE. • Method validation was carried out on FFPE archive blocks from experimental trials. • A RT-qPCR comparative study of FFPE and RNAlater field samples was performed. • The modified assay may overcome the limitations of field sampling at slaughterhouse. In the context of mRNA biomarker profiling, formalin fixed paraffin embedded (FFPE) samples represent an interesting source for retrospective analysis. However, the implementation in routine analysis of FFPE samples, following legislation demands for validated and accredited methods, often requires critical revision and optimization. In the frame of an official control program the validation study of a molecular test for detection of sex steroids administration in calves, based on quantification of progesterone-Receptor mRNA in bulbo-urethral gland samples, was performed: analyses were made on FFPE tissues sections routinary used for histological investigations and compared with RNAlater tissue preservation. To overcome the limitations of original assays several modifications were tested. Obtained results confirmed how Progesterone-Receptor assay represent a useful tool to study suspected cases of sex steroid illicit administration in veal calves, complementary to histological and/or immune histochemical investigation, overcoming the limitation of field studies, where optimal pre-analytical condition cannot always be guarantee. [ABSTRACT FROM AUTHOR]

Abstract Fighting food frauds is a ceaseless challenge because of the constant evolution of fraudulent practices and for the consequences both on consumers' and on globalized trade. In Italy fish is a vulnerable commodity for frauds thanks to the high national production, importation and consumption and it is important to monitor the entire food chain in order to detect and prevent fraudulent actions, such as species substitutions, which is considered the most common fraud in seafood. Aim of this study was to realise a targeted Monitoring Plan to estimate the prevalence of fish species substitutions in Italy. As a first step, Italian fish supply chain, from production to selling, was analysed, in order to identify products and chain points at risk, by reviewing literature and by involving, in two focus groups, food inspectors and representatives of the large scale food distribution system. Then a monitoring plan was designed by sampling three fish species considered at major risk for their economic value and/or large consumption, i.e. tuna, grouper and flat fishes, at different selling points (wholesale markets, retail markets, fish shops and supermarkets). From February to March 2017, 242 samples from fresh, frozen or transformed whole fishes or fillets were sampled in 13 Italian cities (5 in Northern Italy, 2 in Central Italy, 3 in Southern Italy and 3 in the main Islands). Samples were analysed by "FINS" (Forensically Informative Nucleotide Sequencing), using two markers: cytochrome oxidase subunit I gene (COI) as first option and then mitochondrial cytochrome B gene (cytb), if necessary to identify uncertain or unassigned samples. Species substitutions were uncovered in 8.7% of analysed samples, principally related to grouper (prevalence of 14.71%). Supermarkets resulted the selling points with a major number of frauds (prevalence of 12.79%). Substituted species were taxonomically related to those declared on the label and no species harmful for consumers were detected. Results obtained can give to National Authorities a detailed frame of trends in fish substitution frauds in Italy, providing also relevant information to put into effect control measures. Highlights • Targeted Italian Monitoring Plan to estimate the prevalence of fish substitution frauds in Italy. • Results of the first National Monitoring Plans on fish substitutions. • Trends in fish substitution frauds in Italy giving information to put into effect control measures. [ABSTRACT FROM AUTHOR]

Abstract: Asbestos has been banned in many countries, including Italy. However, sources of exposure may still exist, due to asbestos in-situ or past disposal of asbestos-containing waste. In an urban area with past high environmental exposure, like Casale Monferrato, the lung fiber burden in sentinel animals may be useful to identify such sources. A pilot study was conducted to assess the feasibility of its determination in wild rats, a suitable sentinel species never used before for environmental lung asbestos fiber burden studies. Within the framework of pest control campaigns, 11 adult animals from 3 sites in the urban area of Casale Monferrato and 3 control rats from a different, unexposed town were captured. Further, 3 positive and 3 negative control lung samples were obtained from laboratories involved in breeding programs and conducting experimental studies on rats. Tissue fiber concentration was measured by scanning electron microscopy with energy dispersive spectrometry. Asbestos (chrysotile and crocidolite) was identified in the lungs from rats from Casale Monferrato, but not in control rats and in negative control lung samples. Asbestos grunerite at high concentration was found in positive control lung samples. Measurement of the lung fiber burden in wild rats has proved feasible: it was possible not only to detect, but also to characterize asbestos fibers both qualitatively and quantitatively. The pilot study provides the rationale for using wild rats as sentinels of the soil contamination level in Casale Monferrato, to identify areas with the possible presence of previously unrecognized asbestos sources. [Copyright &y& Elsevier]

Abstract: The study was designed to evaluate anti-tumour properties of Iraqi propolis collected from Mosul region (M) on HL-60 and HCT-116 cell lines and on HCT-116 in vivo. M induced an inhibitory effect against the proliferation of HL-60 and colony potential of HCT-116 cells. The apoptosis in HL-60 cells was associated with down-regulation of Bcl-2 and activation of Bax, while in HCT-116 cells, necrotic features were observed; size of cells was dramatically increased by swelling of cytoplasm and loss of membrane integrity, cell rupture and release of cellular contents. Analysis of BrdU/DNA cell cycle in both cell lines showed that M induced cell cycle perturbations in both BrdU positive and BrdU negative cells. The exposure of HL-60 to M caused γ-H2AX in a dose dependent manner and was associated with induction of apoptosis. The experiments in HCT-116 tumor-bearing mice showed that oral administration of propolis at doses that caused no detectable toxicity was associated with a decrease in mitotic cells and an increase in endoreduplications, increased p53 and decreased Ki-67 expression of cells in tumor sections. This study provides the rationale to investigate the potential beneficial effect of propolis in the diet of patients receiving anti-cancer therapies. [Copyright &y& Elsevier]

The olfactory system (OS) is involved in many infectious and neurodegenerative diseases, both human and animal, and it has recently been investigated in regard to transmissible spongiform encephalopathies. Previous assessments of nasal mucosa infection by prions following intracerebral challenge suggested a potential centrifugal spread along the olfactory nerve fibers of the pathological prion protein (PrPSc). Whether the nasal cavity may be a route for centripetal prion infection to the brain has also been experimentally studied. With the present study, we wanted to determine whether prion deposition in the OS occurs also under field conditions and what type of anatomical localization PrPSc might display there. We report here on detection by different techniques of PrPSc in the nasal mucosa and in the OS-related brain areas of sheep affected by natural scrapie. PrPSc was detected in the perineurium of the olfactory nerve bundles in the medial nasal concha and in nasal-associated lymphoid tissue. Olfactory receptor neurons did not show PrPSc immunostaining. PrPSc deposition was found in the brain areas of olfactory fiber projection, chiefly in the olfactory bulb and the olfactory cortex. The prevalent PrPSc deposition patterns were subependymal, perivascular, and submeningeal. This finding, together with the discovery of an intense PrPSc immunostaining in the meningeal layer of the olfactory nerve perineurium, at the border with the subdural space extension surrounding the nerve rootlets, strongly suggests a probable role of cerebrospinal fluid in conveying prion infectivity to the nasal submucosa. [ABSTRACT FROM AUTHOR]

• This paper describe the first in vivo trial supporting bile as matrix for DXM detection at slaughterhouse. • DXM induces severe thymic atrophy in Chianina beef bull identifiable through histology and morphometry. • Results obtained by bile analyses are supported by histological study of thymus. 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. [ABSTRACT FROM AUTHOR]

Reptiles are usually asymptomatic carriers of Salmonella , with the manifestation of typical clinical signs of acute forms in adult and non-immunocompromised animals being considered exceptions. In the present case, an adult male corn snake ( Pantherophis guttatus ) was found dead due to septic shock 48 h after consuming a feeder mouse purchased online. The snake's tissue samples and faeces were cultured for bacteria isolation. Microbiological examinations of the snake and mouse livers revealed the presence of Salmonella enterica subsp . enterica serovar Midway. A whole-genome analysis of these two isolates showed a high correlation between them: they belonged to the strain type ST-357 for the classic MLST scheme and to the strain type ST 171322 for the cgMLST scheme. Also, a virulence gene analysis revealed the presence of stdB and STM3026 genes. This report conveys a case of food-borne salmonellosis in a pet snake, transmitted from a feeder mouse, likely responsible for the snake's death due to septic shock. It highlights the relevance of feeder mice as a source of Salmonella infections in snakes and the associated risks to human health.

The use of anabolic-androgenic steroids (AASs) as growth promoters in farm animals is banned in the European Union, representing both an illicit practice and a risk for consumer health. However, these compounds are still illegally administered, often in the form of synthetic esters. This work aimed to characterize significant coding RNA perturbations related to the illicit administration of testosterone and nandrolone esters in fattening pigs. A total of 27 clinically healthy 90-day-old pigs were randomly assigned to test and control groups. Nine animals were treated with testosterone esters (Sustanon ® ) and other nine with nandrolone esters (Myodine ® ). At the end of the trial, liver samples were collected and analyzed using RNAseq, allowing the identification of 491 differentially expressed genes (DEGs). The transcriptional signature was further characterized by a smaller sub-cluster of 143 DEGs, from which a selection of 16 genes was made. The qPCR analysis confirmed that the identified cluster could still give good discrimination between untreated gilt and barrows compared to the relative testosterone-treated counterparts. A conclusive field survey on 67 liver samples collected from pigs of different breeds and weight categories confirmed, in agreement with testosterone residue profiles, the specificity of selected transcriptional biomarkers, showing their potential applications for screening purposes when AAS treatment is suspected, allowing to focus further investigations of competent authorities and confirmatory analysis where needed.