Thymus and meat physicochemical measurements to discriminate calves treated with anabolic and therapeutic doses of dexamethasone.

To preserve the Europe consumers' health, the use of glucocorticoids as growth promoters is prohibited in cattle fattening. In 2008, the Italian Ministry of Health associated to the official control a national monitoring plan based on the histological thymus analysis to identify animals illegally treated with corticosteroids. However, since corticosteroids are authorized and widely used for therapeutic purposes, it is necessary to verify whether the thymus histological test and some physicochemical traits in meat are able to discriminate doped calves from dexamethasone therapeutic treated ones. The aims of this study were (i) to establish whether the therapeutic and illicit corticosteroid treatments of calves could be differentiated through histological evaluation of thymus and by physicochemical meat traits; (ii) to identify a restricted number of physicochemical traits that could differentiate dexamethasone treated from untreated calves. Three groups of 15 calves each were included in this study: group dexamethasone therapeutic treatment treated with dexamethasone 21-phosphate disodium salt at a therapeutic dose (2 mg/kg of live weight for three consecutive days); group dexamethasone anabolic treatment orally treated with dexamethasone 21-phosphate disodium salt according to a presumed anabolic protocol (0.4 mg/day per animal for 20 days); group placebo control treated with a placebo served as control. Results demonstrated that groups could be easily discriminated by thymus microscopy as well as by two meat markers, namely, cooking loss and shear firmness or Warner-Bratzler shear force. The combination of thymus microscopic features and meat physicochemical traits could be used as a practical, economic and accurate screening strategy to discriminate between meat from illegally and therapeutically treated calves. This new reliable and simple tool could contribute to identify animals treated with dexamethasone in those countries where glucocorticoids are illegally used as growth promoters. More in general, this system could be included in the framework of official controls, and applied to verify suppliers' reliability by the meat industry.

Spatial asymmetry of the paternity success in nests of a fish with alternative reproductive tactics.

Guard-sneaker tactics are widespread among fish, where territorial males defend a nest and provide parental care while sneakers try to steal fertilizations. Territorials and sneakers adopt diverse pre- and post-mating strategies, adjusting their ejaculate investment and/or behavioural responses to the presence of competitors. The relative distance of competitors from the spawning female plays a major role in influencing male mating strategies and the resulting paternity share. However, territorial male quality and sneaking intensity do not fully account for the variability in the relative siring success occurring among species. An often neglected factor potentially affecting sneakers proximity to females is the nest structure. We conducted a field experiment using the black goby, whose nests show two openings of different size. We found that territorial males defend more and sneaking pressure is higher at the front, larger access of the nest than at the back, smaller one. Moreover, microsatellite paternity analysis shows that territorials sire more offspring at the back of their nest. Such a predictable spatial distribution of the paternity share suggests that nest structure might work as an indirect cue of male relative siring success, potentially influencing the territorial male investment in parental care and/or the female egg deposition strategy.

Wild rats as urban detectives for latent sources of asbestos contamination.

Based on a large body of evidence asbestos minerals have been classified as carcinogens. Despite the Italian ban on asbestos in 1992 and the subsequent remediation activities, latent sources of contamination may still represent a hazard where asbestos were particularly used. Using wild rats as sentinel animals, this study aimed at uncovering sites with the greatest potential for non-occupational exposure to asbestos in the city of Casale Monferrato (Piedmont Region, Italy), where the largest Italian manufacturing plant of asbestos-cement had been active. During the study period (2013-2015) a total of 40 wild rats were captured from 16 sampling capture points. The lungs of wild rats have been investigated by using scanning electron microscopy (SEM) with energy dispersive spectroscopy (EDS). The SEM-EDS detected the presence of asbestos fibers (tremolite/actinolite, amosite, and chrysotile) in rats' lungs from 11 sampling points. The hypothetical rats' home-range and the observed site-specific concentration of asbestos fibers per gram of dry lung tissue were used to identify areas to be targeted by additional search of latent sources of asbestos. In conclusion, our results showed that the use of wild rats as sentinel animals may effectively integrate the strategies currently in use to reduce the exposure to asbestos.

Impact of cadmium exposure on swine enterocytes.

We tested cadmium (Cd2+) effects on porcine IPEC-J2 cells, which represent an in vitro model of the interaction between intestinal cells and both infectious and non-infectious stressors. Accordingly, we investigated the effects of low (2 µM) to moderate (20 µM) concentrations of Cd2+, in terms of pro-inflammatory gene expression and protein release, as well as of infectivity in a Salmonella typhimurium penetration model. Our data showed a significant (P < .001) increase of intracellular Cd2+ after 3, 6 and 24 h of exposure with respect to levels at 1 h. These data showed the ability of IPEC-J2 to absorb Cd2+ as a function of both time and concentration. Also, the absorption of this heavy metal was related to a significant modulation of important pro-inflammatory messengers. In particular, down-regulation of IL-8 was associated with a significant decrease of Salmonella typhimurium ability to penetrate into IPEC-J2 cells, in agreement with a previous study in which an anti-IL 8 antibody could significantly inhibit Salmonella penetration into the same cells (Razzuoli et al., 2017). This finding demonstrates the ability of Cd2+ to affect the outcome of an important host-pathogen relationship. In conclusion, our study highlighted the ability of an environmental pollutant like Cd2+ to modulate innate immune responses in terms of chemokine release and gene expression, and susceptibility to microbial infections.

First description of a mortality event in adult Pacific oysters in Italy associated with infection by a Tenacibaculum soleae strain.

Summer mortality episodes in adult Pacific oysters have been described since the 1950s in various farming areas. Starting in 2012, a recrudescence of mortalities in commercial-sized oysters was first observed in France and then in Italy, with seasonality extension and translation later in the year. Moribund individuals collected during an event in Italy in December 2014 showed yellowish lesions of the mantle and adductor muscle. Histological examination revealed filamentous bacteria associated with necrotic areas. Quantitative PCRs targeting OsHV-1 and Vibrio aestuarianus detected only high loads of the pathogenic bacteria in tissues of symptomatic individuals. A lower diversity of the hemolymph microbiota was also evidenced in moribund individuals, with a predominance of Vibrio and Arcobacter species. A strain of Flavobacteriaceae was isolated from all the symptomatic individuals. Sequence analysis of the 16S rRNA gene identified the strain as Tenacibaculum soleae. When strain pathogenicity was tested by injection in adult individuals, it induced mortality rates of up to 45%, even in the absence of V. aestuarianus. As mortality occurred only 11 days post-infection, further investigation is needed to determine its effective virulence in natural conditions. This is the first description of a Tenacibaculum strain associated with bivalve mortalities.

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 2.0 ng mL(-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 6ß-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 1.1 ng mL(-1). Similar prednisolone levels were found in urine samples of untreated calves. Moreover the presence of 6ß-hydroxyhydrocortisone below the CCα value made possible to estimate the 6ß-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.

Development, validation and application to real samples of a multiresidue LC-MS/MS method for determination of ß2 -agonists and anabolic steroids in bovine hair.

ß(2) -agonists are often abused in cattle breeding because of their effects on animal growth and meat properties. The use of ß(2) -agonists as growth promoters is forbidden in the European Union (Council Directive 96/23/EC classifies them into group A of Annex I), due to their toxicity and carcinogenic properties, as for anabolic steroids, which are often administered in combination with ß(2) -agonists, to promote the storage of proteins and increase muscle size. A unique confirmatory liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the quantitative detection of 13 ß(2) -agonists and anabolic steroids plus the qualitative identification of other three analytes in bovine hair was developed and validated, according to Decision 2002/657/CE. Hair samples were washed with dichloromethane, digested within a NaOH solution and subjected to liquid-liquid extraction. The analysis was performed by high performance liquid chromatography coupled to a triple quadrupole mass spectrometer operating in the selected reaction monitoring mode. The absence of matrix interferents, together with good repeatability of both retention times and relative abundances of diagnostic transitions, allowed the correct identification of all analytes. The quantitative calibrations obtained from spiked blank hair samples proved linear in the range tested. CCα and CCß ranged from 0.5 ng/g to 30 ng/g. Intralaboratory reproducibility (CV%) ranged between 5.0 and 17.7 and trueness between 96% ± 7% and 105% ± 8%. The applicability of the method to real positive samples was demonstrated for both ß(2) -agonists and anabolic steroids. 17α-boldenone was found in most (70%) hair samples obtained from untreated animals, supporting the hypothesis of endogenous production of this steroid.

Identification by a proteomic approach of a plasma protein as a possible biomarker of illicit dexamethasone treatment in veal calves.

Corticosteroids have become the most widespread illegal growth promoters in veal calves and beef cattle. Testing for corticosteroids relies on either direct detection of compounds or their metabolites or indirect detection to identify changes in biological pathways. We used a comparative proteomic approach, based on two-dimensional electrophoresis (2DE), to identify plasma protein markers after short-term dexamethasone administration in veal calves. Twenty-three male Friesian veal calves were treated experimentally with dexamethasone sodium phosphate: 10 received low-dose administration of the drug (0.4 mg day⁻¹ per os) for 20 consecutive days (treatment group); 10 received the drug at therapeutic dosage (2-4 mg kg⁻¹ i.m.) for 3 consecutive days (comparison group). Three animals were not treated (control group). Plasma samples were collected from each animal at six time points (T1-T6; treatment and control group) and at four time points (T1-T4; comparison group) and stored at -80°C until analysis. Plasma proteins were quantified and analysed in triplicate by 2DE. The images were analysed with Bionumerics® software. Comparison of 2DE maps obtained from blood samples at T1 (before treatment) and at T6 (final sampling) showed a significant disappearance (p < 0.001) of two protein spots at T6 in the treatment group. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis and immunoblotting identified these isoforms as serum paraoxonase/arylesterase 1 precursor (PON1). Synthesised in the liver and released into the blood, PON1 has an important role in lipid metabolism. The absence of variation of this protein in the comparison group suggests that the marker has good specificity for detecting illicit corticosteroid treatment.

Granulomatous lesions in a wild mullet population from the eastern Ligurian Sea (Italy): mycobacteriosis vs. pseudotuberculosis.

Mycobacterium spp. and Photobacterium damselae subsp. piscicida are recognized as the most frequent causative agents of granulomatous lesions in fish. Although frequent episodes of mycobacterial infections have been reported in wild fish worldwide, only sporadic cases have been documented to date in Italy. To investigate for the presence of lesions referable to mycobacteriosis and to identify the mycobacterial species involved, a total of 159 wild mullets were fished from the eastern coast of the Ligurian Sea, killed and necropsied. Liver and spleen samples were collected from all fish for histopathological and microbiological analyses. Molecular investigations for identification of Photobacterium damselae subsp. piscicida were performed. Gross examination revealed granulomatous lesions in one animal; microscopically, 42.14% of fish displayed granulomas with various histological features, 19.50% resulted positive at Ziehl-Neelsen staining, and were confirmed as mycobacterial lesions by culture. The identified colonies were characterized as M. fortuitum, M. abscessus, M. flavescens, M. chelonae, M. septicum and M. nonchromogenicum. In all, 35% of animals resulted positive for Photobacterium damselae subsp. piscicida. These data suggest widespread mycobacterial infection also by Photobacterium damselae subsp. piscicida infections in wild fish. Moreover, the pathogenicity of some mycobacterial species, previously considered as saprophytic, was demonstrated.

Histopathology as a simple and reliable method to detect 17ß-oestradiol illegal treatment in male calves.

17ß-Oestradiol is a steroid hormone banned as a growth promoter in food-producing animals all over Europe because of its carcinogenicity. Despite mandatory monitoring of illegal treatment all over Europe, official analytical methods in use test negative a few days after 17ß-oestradiol administration, requiring new sensitive tools to ensure a high level of protection for consumers. The aim of this work was the evaluation of the accuracy of histopathology and immunohistochemistry for progesterone receptor (PR) as a screening method for the detection of low-dosage illegal treatments with 17ß-oestradiol. Fresian male calves (153) were farmed under controlled conditions, and 89 of them were treated with 17ß-oestradiol (5 mg/animal once a week for 4 weeks). After 15 days of suspension, all animals were slaughtered and sexual accessory glands (prostate and bulbo-urethral glands) were sampled for histological examination and immunohistochemical staining with anti-PR antibody (clone hPRa 2). Microscopically 86 out of 89 bulbo-urethral glands showed mild to severe metaplasia, while mild metaplasia was observed only in 1 control. Eighteen out of 89 samples of prostate did not show metaplastic lesions. Immunopositivity for PR characterised all treated animals, while no signal was detected in controls. These findings show that metaplasia of the sexual accessory glands is a sensitive and specific parameter for illegal 17ß-oestradiol treatment in calves at the slaughterhouse, while the appliance of immunohistochemistry for PR can improve to 100% the accuracy of this highly reliable histological approach.

Application of absolute qPCR as a screening method to detect illicit 17ß-oestradiol administration in male cattle.

It has been previously demonstrated that the progesterone receptor gene is up-regulated in the sex accessory glands of pre-pubertal and adult male bovines after 17ß-oestradiol treatment. In the present study, a qualitative screening method was optimised to detect 17ß-oestradiol treatment using absolute quantification by qPCR of the progesterone receptor gene to determine the amount of gene expression in bulbo-urethral glands. An external standard curve was generated and developed with TaqMan® technology. Based on two in vivo experiments, the decision limit CCα, sensitivity and specificity of this screening method were established. Trial 1 consisted of 32 Friesian veal calves divided into two groups: group A (n = 12), consisting of animals treated with four doses of 17ß-oestradiol (5 mg week(-1) per animal); and group B (n = 20), consisting of control animals. Trial 2 was performed on 26 Charolaise beef cattle that either received five doses of 17ß-oestradiol (group C; 20 mg week(-1) per animal; n = 6) or remained untreated (group D; n = 20). Further, progesterone receptor gene expression was evaluated in beef and veal calves for human consumption. A specific CCα on 20 Piedmontese control beef cattle was calculated to include these animals in a field investigation. Five out of 190 beef cattle and 26 out of 177 calves tested expressed the progesterone receptor gene above their respective CCα and they were classified as being suspected of 17ß-oestradiol treatment. Additionally, 58% of veal calves that tested suspect via qPCR exhibited histological lesions of the bulbo-urethral gland tissue, which are typical of oestrogen administration and are consistent with hyperplasia and metaplasia of the glandular epithelium.

Histology as a valid and reliable tool to differentiate fresh from frozen-thawed fish.

Selling fish products as fresh when they have actually been frozen and thawed is a common fraudulent practice in seafood retailing. Unlike fish products frozen to protect them against degenerative changes during transportation and to extend the product's storage life, fish intended for raw consumption in European countries must be previously frozen at -20° C for at least 24 h to kill parasites. The aim of this study was to use histological analysis to distinguish between fresh and frozen-thawed fish and to evaluate this method for use as a routine screening technique in compliance with the requirements of European Commission Regulation No. 882/2004 on official food and feed controls. Method performance (i.e., accuracy and precision) was evaluated on tissue samples from three common Mediterranean fish species; the evaluation was subsequently extended to include samples from 35 fish species in a second experiment to test for method robustness. Method accuracy was tested by comparing histological results against a "gold standard" obtained from the analysis of frozen and unfrozen fish samples prepared for the study. Method precision was evaluated according to interrater agreement (i.e., three laboratories with expertise in histopathology in the first experiment and three expert analysts in the second experiment) by estimating Cohen's kappa (and corresponding 95 % confidence intervals) for each pair of laboratories and experts and the combined Cohen's kappa for all three experts and laboratories. The observed interrater agreement among the three laboratories and the three experts indicated high levels of method accuracy and precision (high sensitivity and specificity) and method reproducibility. Our results suggest that histology is a rapid, simple, and highly accurate method for distinguishing between fresh and frozen-thawed fish, regardless of the fish species analyzed.

Development of tertiary lymphoid structures in the kidneys of pigs with chronic leptospiral nephritis.

Tertiary lymphoid organs (TLOs) are structures that are morphologically and functionally similar to secondary lymphoid organs. TLOs usually arise in a background of chronic inflammation. Several histological patterns of interstitial nephritis have been documented in porcine leptospirosis. Among them the lympho-follicular pattern is characterized by infiltrates of mononuclear cells organized in lymphoid follicle-like structures. Immunohistological analysis of 5 cases of porcine lympho-follicular nephritis associated with Leptospira Pomona infection demonstrated the presence of inflammatory cell populations, including B cells, T cells, macrophages and follicular dendritic cells (FDCs), which were compartmentalized as in TLOs. Immunohistochemistry for Leptospira Pomona revealed an intimate association between leptospiral antigen and FDCs. Overexpression of MHCII in different populations of both professional and non-professional antigen presenting cells was also demonstrated. FDCs play role during TLOs induction for their ability to retain non-self antigens in the form of immune complexes, thus causing persistent T cell activation, generation of a complex cytokine network and stimulation of humoral immunity. Sustained bacterial antigen presentation in the context of chronic leptospiral nephritis, may also lead to autoimmune mechanisms involved in the generation of TLOs. Whether lymphoid neogenesis and TLOs play a protective role in porcine leptospiral nephritis is still unclear.

Development of an enhanced histopathological approach to detect low-dose dexamethasone illicit treatment in veal calves.

Dexamethasone is one of a number of synthetic corticosteroids illegally used to promote growth in food-producing animals. Since these low-level drug cocktails evade detection by currently available chemical methods, simple biological indicators that can aid in laboratory analysis are needed. In an attempt to devise an accurate biological method that could detect illicit drug treatment in food-producing animals, we characterized microscopic morphologic alterations of the thymus in veal calves administered low-dose dexamethasone versus control animals. For this purpose, 122 male calves were farmed for 6 months in controlled condition: 81 animals were orally administered dexamethasone (0.4 mg day(-1)) for 20 days during the sixth month and the remaining 41 were kept as control. Urine samples were collected systematically during the treatment period, the suspension period and at the slaughterhouse. All animals were slaughtered 10 per day starting from 10 days after the last dexamethasone administration and the thymus was sampled for histological examination. The difference between the two animal groups was evaluated by means of a non-parametric test of hypothesis. No residues were detected in the urines collected since the third day after the last administration, whereas morphometric analysis of the thoracic thymus revealed a significant decrease in the cortex:medulla ratio in the treated animals (p<0.0005). We can conclude that this histological approach offers encouraging prospects as a screening method to overcome current limitations in controlling growth promoter abuse.

Cerebral toxoplasmosis in striped dolphins (Stenella coeruleoalba) stranded along the Ligurian Sea coast of Italy.

This article reports the results of necropsy, parasitologic, microbiologic, histopathologic, immunohistochemical, indirect immunofluorescence, biomolecular, and serologic investigations on 8 striped dolphins (Stenella coeruleoalba) found stranded from August to December 2007 on the Ligurian Sea coast of Italy. Severe, nonsuppurative meningoencephalitis was found in 4 animals, as characterized by prominent perivascular mononuclear cell cuffing and macrophage accumulations in neuropil. These lesions were associated with mild lymphocytic-plasmacytic infiltration of choroid plexuses in 1 dolphin. Toxoplasma gondii cysts and zoites, confirmed by immunohistochemical labeling, were scattered throughout the brain parenchyma of 2 of the 4 dolphins. No viral inclusions were seen in the brain of any animal. Other findings included severe bronchointerstitial pneumonia and pulmonary atelectasis, consolidation, and emphysema. Parasites were identified in a variety of organs, including lung (Halocerchus lagenorhynchi). Microbiologic and serologic examinations for Brucella spp were negative on all 8 dolphins. The 4 animals with meningoencephalitis had serum antibodies against T gondii (titers ranging from 1:80 to 1:320) but not against morbillivirus. In contrast, the other 4 dolphins were seropositive for morbillivirus (with titers ranging from 1:10 to 1:40) but seronegative for T gondii. No morbillivirus antigen or nucleic acid was detected in the tissues of any dolphin. It is concluded that the severe lung and brain lesions were the cause of death and that T gondii was the likely etiologic agent of the cerebral lesions. Morbillivirus infection was not considered to have contributed to death of these animals.

Interlaboratory trial on TSE rapid tests for the control of the Italian scrapie surveillance network.

Scrapie, a neurodegenerative disease of sheep and goats and one of several transmissible spongiform encephalopathies (TSEs) has been subject to mandatory active surveillance in EU through rapid testing since 2002. Regulation EC/999/2001 on TSE surveillance requires that each Member State's National Reference Laboratory for TSE periodically verifies diagnostic standards and methods by comparative testing. In 2007 the Italian Reference Centre carried out the first ring trial for classical scrapie on a set of 21 negative and 9 positive homogenated brainstems, the latter consisting of three replicates of an ARQ/ARQ scrapie positive sample diluted 1:10, 1:20 and 1:50. The purpose of the study was to verify the interlaboratory agreement in term of Cohen's kappa (k) of the rapid systems currently used by the 25 national rapid test laboratories (RTLs) [laboratories: Biorad TeSeE (17 laboratories), Enfer TSE version 2.0 (4 laboratories), Idexx Herd chek BSE-scrapie antigen kit (3 laboratories) and Prionics check LIA SR (1 laboratory)]. Our results show that the agreement among the laboratories using the same rapid test varied between 0.84 and 1, while the estimated overall agreement among the 25 laboratories was very good (k-combined=0.87, 95% confidence interval 0.85-0.89). Nevertheless, as regards differences in analytical sensitivity among the rapid tests in use, under-recognition of pre-clinical scrapie cases by lower performing systems must be expected.

Evaluation of an enzyme immunoassay for the detection of central nervous system tissue contamination at the slaughterhouse.

To protect public health from bovine spongiform encephalopathy, European Commission Regulation EC 1139/2003 on monitoring programs and specified risk material requires that as of 1 October 2003, each member state has in place a sampling plan with an appropriate laboratory test to detect central nervous system (CNS) tissue in bovine head meat harvested at slaughterhouses or cutting plants. With this study, we wanted to evaluate the accuracy and reliability of an enzyme immunoassay, the RIDASCREEN Risk Material 10/5, in targeting a CNS-specific marker, the glial fibrillary acidic protein. A receiver operating characteristics curve was plotted to identify the best cutoff of CNS concentration. Reliability was calculated by Cohen's kappa on data from two diagnostic sessions. Test performance showed high sensitivity and specificity (97.9 and 97.4%, respectively) for a cutoff value between positive and negative at a CNS concentration of 0.049%; reliability of test precision was also very good. When these criteria are applied, the RIDASCREEN Risk Material 10/5 test appears to be a reliable tool for monitoring CNS tissue contamination in meat. This diagnostic procedure should therefore be recommended for national application in monitoring programs.

A case of scrapie in a sheep carrying the lysine-171 allele of the prion protein gene.

Susceptibility to scrapie in sheep depends on the host PrP genotype. No data about the linkage of the rare ARK allele to differential scrapie susceptibility are currently available. Several tissues isolated from sheep from an Italian scrapie outbreak and carrying the ARK allele were examined for the presence of the pathological prion protein. A weak positivity was detected only by Western blot in the brainstem of one ARK/ARH sheep. This result shows that the ARK allele does not confer full resistance against scrapie and that the allele needs to be studied further before it can be considered for breeding purposes.