Marzia Pezzolato

Studies on the presence of natural and synthetic corticosteroids in bovine urine.

Natural and synthetic corticosteroids are widely used in veterinary medicine for their anti-inflammatory properties, but are also illegally used in animal breeding as growth-promoting agents: this latter application in livestock production has been banned within the European Union due to health concerns for the consumer. In this work urine samples collected from bovines experimentally treated with dexamethasone (0.4 mg of dexamethasone 21-disodium phosphate per capita/day for 20 consecutive days) and bovines bred under strictly controlled conditions were investigated for the presence of natural and synthetic corticosteroids, using a simple multi-residue liquid chromatography-tandem mass spectrometry method, developed and validated in accordance with the criteria of the Commission Decision 2002/657/EC. The aim of this work is to investigate the effect of a low dosage and long term dexamethasone treatment on the levels of endogenous corticosteroids in cattle and to evaluate the possible presence of prednisolone residues in bovines bred under strictly controlled conditions. Our findings confirm the high and rapid rate of dexamethasone urinary excretion. Dexamethasone treatment elicited an early reduction of hydrocortisone and cortisone, suggesting the disappearance of these two hormones as an indirect indicator of corticosteroid treatment in cattle. Prednisolone residues were found (concentration interval 0.4-1.4 ngmL(-1)) in urine samples collected from control bovines especially at the slaughterhouse, together with high levels of hydrocortisone and cortisone. Further studies are necessary to find out the reason of unexplained excretion of this hormone in urine samples of untreated bovines.

Olfactory System Involvement in Natural Scrapie Disease

ABSTRACT 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.

Comparison of histologic techniques for the diagnosis of bovine tuberculosis in the framework of eradication programs

Rapid diagnosis of tuberculosis in cattle reacting positive in antemortem assays is crucial in countries where eradication programs are operated to confirm the presence of the infection in tuberculosis-free herds. This study evaluated the accuracy of histopathologic examination by hematoxylin and eosin and Ziehl-Neelsen (ZN) staining applied in this framework, when suspected lesions are caused by low infectious doses and are detected in early stages of the disease. For this purpose, histologic methods were compared with mycobacterial culture as reference test on suspected lymph node samples from 173 cattle reacting positive in antemortem tests. Histopathology demonstrated high sensitivity (93.4%) and specificity (92.3%), while ZN sensitivity and specificity were respectively 33.9% and 100%. There was good agreement between histopathology and bacterial culture, suggesting that histopathologic examination is a reliable tool for rapid diagnosis in countries where active tuberculosis eradication programs allow the prompt identification and elimination of reactor cattle. Histopathology permits identification of typical mycobacterial lesions and its differentiation from other causes.

Immunodetection of 17β-estradiol in serum at ppt level by microcantilever resonators

To date control strategies in detecting anabolic agents for promoting growth of food producing animals are mainly related to screening techniques based on immunochemical and physiochemical methods, whose major limit is represented by relative low analytical sensitivity. As a consequence, consumers are currently exposed to molecules with potential carcinogenic effects such as 17β-estradiol, the most powerful substance with estrogenic effect. Therefore, high analytical sensitivity screening and confirmatory methods are required, coupling easiness of use and efficiency. We here report on the immunodetection of 17β-estradiol in serum by antibody-immobilized microcantilever resonators, an innovative biosensing platform able to quantify an adsorbed target mass (such as cells, nucleic acids, biomolecules, etc.) thanks to a shift in resonance frequency. Our tool based on microcantilever resonator arrays has shown to be capable of discriminating treated and untreated animals, showing the ability of detecting traces of 17β-estradiol in serum at concentrations lower than the present accepted physiological serum concentration threshold value (40 ppt) and commercial ELISA tests (25 ppt). The method exhibits a limit of detection of 20 ppt and a limited cross-reactivity with high concentrations (10 ppb) of similar molecules (testosterone).

Excretion profile of corticosteroids in bovine urine compared with tissue residues after therapeutic and growth-promoting administration of dexamethasone.

The illicit use of dexamethasone as growth-promoting agent in animal breeding is still practiced within the EU constituting a health risk for meat consumers. An experimental study was developed to assess dexamethasone urinary excretion and tissue distribution (liver, kidney, and muscle) in male calves after therapeutic and growth-promoting administration. Urine and tissue samples collected from treated and untreated bovines were also investigated for the presence of other natural and synthetic corticosteroids (prednisolone, prednisone, hydrocortisone, and cortisone), in order to study a possible correlation with dexamethasone administration and to clarify prednisolone origin. Analyses were performed by a multi-residue LC-MS/MS method developed and validated according to the Commission Decision 2002/657/EC. The results confirm the rapid rate of dexamethasone urinary excretion, irrespective of the dosage, the duration and the route of administration, and the disappearance of cortisone and hydrocortisone during the treatment. Dexamethasone was distributed to the tissues where the elimination rate proceeded relatively slower as suggested by the presence of residues one month after the withdrawal of the therapeutic treatment. An increase in the number of positive findings for prednisolone, in association with higher levels of cortisone and hydrocortisone, was observed in urine samples collected from slaughterhouse rather than those collected at the farm. Prednisone residues were found only in one urine sample that showed the highest levels of prednisolone, hydrocortisone, and cortisone. The occurrence of prednisolone residues in urine and even in tissue samples confirms the endogenous nature of this molecule.

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.

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.

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

17β-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 17β-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 17β-oestradiol. Fresian male calves (153) were farmed under controlled conditions, and 89 of them were treated with 17β-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 17β-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.

The wild rat as sentinel animal in the environmental risk assessment of asbestos pollution: A pilot study

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.

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 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 1.1 ng mL(-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.