The assurance of safe and good quality food is a fundamental request of the consumers. Dexamethasone and other corticosteroids are frequently illegally used as growth-promoters in livestock production [6
], often administered at low dosage alone or combined with β-agonists and/or anabolic steroids.
In our study, the results of the chemical analysis in urine and liver samples obtained at the slaughterhouse, confirmed that when DXM is administered at low dosage, its rapid metabolism and excretion makes the residues determination difficult, as the drug is readily depleted after the suspension of the treatment [20
]. In fact, while in group B DXM residues were still detectable and identifiable in all liver samples, in animals of group C, which were treated with lower dosage of DXM and had a 8-day withdrawal period, DXM residues (>CCα) were not detectable in liver of 6 out of 8 animals; moreover, DXM residues detected in 2 liver samples were lower than the MRL = 2 μg/Kg in liver (Commission Regulation (EU) No 37/2010) [5
]. Furthermore, the urine samples collected at the slaughterhouse, had DXM concentrations largely below the CCα in 9 animals of group B, as well as in all the animals of group C. These data are in accordance with the findings of a previously published paper [15
] evaluating DXM urine excretion in beef cattle treated with low-dose of DXM during the drug administration period, as well as 6 days after the end of the treatment. DXM residues were detected in all samples collected during the treatment, although at low concentration levels, and were not detectable anymore 6 days after treatment suspension [15
]. Moreover, a recent study [22
] evaluated drug residue levels in urine and feces, as well as its fixation in bovine hair following a single administration of 0.15 mg/kg b.w. dexamethasone acetate and 0.12 mg/kg b.w. dexamethasone sodium phosphate, by different analytical methods based on GC-MS or LC-MS/MS; the results confirmed the high and rapid urinary excretion rate of DXM, with a maximal concentration measured one day after administration and 98% elimination within 3 days [22
Thus, novel screening tools are needed to accurately reveal the illegal administration of corticosteroids by the meat producing industry.
Our findings demonstrate that low dosages of DXM, administered alone or in association with clenbuterol as growth promoter in beef cattle, according to the protocols often illegally adopted in farm practice [7
], induce morphologic changes in the thymus, resulting in increase fat infiltration with concurrent cortical atrophy. The role of thymus atrophy as an indirect biomarker of corticosteroid administration in beef cattle [14
], as well as in veal calf [7
] has been previously highlighted. However, the reliability of this method could be affected by several factors, such as infectious diseases, intoxication and stress [24
]. In addition, in beef cattle it is rather difficult to distinguish between physiological involution and treatment-induced atrophy, and the thymus score could be influenced by subjective inter-observer variations. Our data indicate that the severity of the histological alteration correlates with the administered DXM dosages, as previously observed [25
]. In fact, among animals treated with lower doses of DXM in association with a β-agonist (group C), thymus atrophy was less evident than in group B, and many thymus samples were scored 2. Samples with thymus score 2 are not discriminative, since they occur in both control and treated animals. The association of more objective parameters as indirect indicators may be helpful in enhancing the sensitivity and specificity of the histological method. Administration of synthetic glucocorticoids, such as DXM, can cause acute thymus involution and have been used as a model system [26
]. Acute thymic involution is characterized by reduction in thymus size caused by acute loss of cortical thymocytes and reduced output of native T cells to the periphery [28
]. Differently, age-induced involution is characterized by a gradual expansion of perivascular spaces and reduction of thymic epithelial spaces capable of supporting thymopoiesis [24
]. Other possible causes of thymus atrophy as autoimmune phenomenon and immunodeficiency syndromes have been shown to affect cellular density with symmetrical reduction of thymic cortical and medullary areas as well [29
]. Our results indicate that the cortex/medulla ratio is a simple and objective parameter, useful for the detection of illegal treatment with low doses of DXM, which could be applied independently from the thymus score. In fact, the C/M value was strongly associated with corticosteroids treatments, with both the protocols applied. The cut off value of 0.93 for the C/M resulted to be highly effective to distinguish control from treated animals with thymus score 2. Furthermore, the C/M value resulted to be not significantly associated with the thymus score. This means that animals with different rates of fat tissue infiltration, due to age-related involution or other factors, could be categorized in treated or untreated group on the basis of the C/M value. In our model, considering 5 lobules randomly selected in each thymus sample, animals with thymus score 2 and at least 2 lobules having C/M < 0.93, should be considered as treated with corticosteroids, with a PPV and NPV both equal to 100%. It should be emphasized that the proposed cut-off value for the C/M has to be validated by studies on a larger population, including different breed and ages of the animals. A similar approach were applied in a recent study on veal calves, treated with low-doses of DXM for 20 days [19
]. Results of the study showed that fat infiltration was not significantly associated with steroidal treatment, while a significant reduction in the C/M was observed in treated animals vs controls [19
]. The findings of both studies seem indicate that the C/M is strongly related to steroidal treatment in both veal calves and beef cattle, being more reliable than the evaluation of fat infiltration in thymus parenchyma. Assessment of the C/M, once validated in a larger population sample, taking in consideration the repeatability and the accordance between different diagnostic laboratories, could represent a promising and standardizable method for screening purposes, to evaluate the diffusion of corticosteroids illicit treatments in beef production.
In our study, the serum cortisol concentration of control and treated animals was evaluated during the rearing period, as well as at the slaughterhouse. Non treated animals (group A), showed high variability in serum cortisol concentration during the whole study period, with higher median values at T0 and at the slaughterhouse, imputable to physiological stress response to handling and transport procedures [30
]. The animals treated with DXM showed inhibition of cortisol secretion during the treatment period, as well as at the slaughterhouse, 3 days after treatment suspension. In fact, DXM acts similarly as endogenous cortisol and inhibits hypothalamus and hypophysis activities exerting a negative effect on ACTH secretion [32
]. These data are in accordance with the findings of a previous experimental study in beef cattle [34
], as well as with those of a field study investigating the thymus morphology and serum cortisol concentration in regularly slaughtered beef cattle [14
]. In the latter study the animals considered to be negative on the basis of thymus morphology had a significantly higher mean cortisol level than those suspected of corticosteroid treatment [14
Animals of group C, which were treated with lower doses of DXM in association with clenbuterol, showed inhibition of cortisol secretion during the treatment period, but serum cortisol concentration was restored to physiological levels at slaughterhouse, 8 days after treatment suspension, indicating that the withdrawal period could affect the reliability of the method.