GR CALUX assay detects synthetic glucocorticoids in calf urine: a validation study.

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 ß 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.

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

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

EU-approved rapid tests might underestimate bovine spongiform encephalopathy infection in goats.

We report the diagnostic sensitivity of 3 EU-approved rapid tests (ELISAs; 1 from IDEXX and 2 from Bio-Rad) for the detection of transmissible spongiform encephalopathy diseases in goats. Ninety-eight goat brainstem samples were tested. All the rapid tests had 100% specificity and ≥80% sensitivity, with the IDEXX test significantly more sensitive than the 2 Bio-Rad tests. All tests detected 100% of samples from goats with clinical scrapie, but missed 8% (IDEXX) to 33% (Bio-Rad SG) of samples from preclinical goats. Importantly, only IDEXX picked up all samples from clinical bovine spongiform encephalopathy (BSE)-infected goats, whereas the other 2 rapid tests missed 15% (Bio-Rad SG) to 25% (Bio-Rad SAP). These results show that a fraction of preclinical scrapie infections are likely missed by EU surveillance, with sensitivity of detection strongly dependent on the choice of the rapid test. Moreover, a significant proportion of clinical BSE infections are underestimated by using either Bio-Rad test. Assuming that the same sensitivity on preclinical goats would also occur in BSE-infected goats, our data suggest that IDEXX is likely the most sensitive test for detecting preclinical field cases of BSE infection in goats, although with an 8% failure rate. These results raise some concerns about the reliability of current EU surveillance figures on BSE infection in goats.

Confirmation of the progesterone receptor as an efficient marker of treatment with 17ß-estradiol in veal calves.

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 (17ß-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 17ß-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 17ß-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 17ß-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.

Effect-based detection of synthetic glucocorticoids in bovine urine.

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.

Immunodetection of 17ß-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 17ß-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 17ß-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 17ß-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).