Effects of an illicit cocktail on serum immunoglobulins, lymphocyte proliferation and cytokine gene expression in veal calves: preliminary results.

Introduction. The immune system (IS) is a complex multicomponent system that comprises lymphoid organs and interacting, specialised cells. Several class of harmful compounds and/or drugs (i.e. organotins, cyclosporine A, dioxins) exert their main toxic actions upon the IS, resulting in immunotoxicity or immune suppression (1). Glucocorticoids (i.e. dexamethasone, DEX) are often illegally used, alone or in combination with steroids or β-agonists, as growth promoters (GPs) in cattle (2). The use of DEX as GP has been associated with thymus atrophy, leucopoenia and decreased lymphocyte proliferative response (3). Besides, it is well known that some other GPs (i.e. clenbuterol, CLEN; 17β-oestradiol, 17βE) markedly influence the IS (4-6). Therefore, the aim of the present study was to describe possible alterations of the IS functions in veal calves administered with an illicit cocktail, by using well known methodologies (lymphocyte proliferation assay, serum immuglobulin concentrations, cytokine gene expression). Materials and Methods. Twelve cross-bred male veal calves were divided in 2 experimental groups, of 6 animals each. The first group (weighing 162 ± 18 kg) was used as control (CTRL), whereas the second one (TRT, 166 ± 19 kg) received 17βE (3 im injections of 10 mg at 17 days intervals), CLEN (20 mg kg-1 dissolved in milk for 40 days) and DEX (4 g per animal for 7 days and then 5 g per animal for a further 5 days, dissolved in milk) for a total of 55 days. Blood sampling were withdrawn at the beginning (T0) and, then, after 15 (T1), 34 (T2), 48 (T3) days and the day before slaughtering (T4). Lymphocytes proliferation assay was executed according to Biolatti et al (2005: 3), by using concanavalin A, pokeweed mitogen and phythaemagglutinin as mitogens. Serum immunoglobulin G (IgG) and M (IgM) concentrations were determined by radial diffusion assay (Bethyl Lab., Montgomery, AL, USA). The effect of treatment upon the cytokine interleukin 1β, interleukin 8 (IL-8), tumour necrosis factor α and interferon γ gene expression levels was evaluated, using the mRNA extracted from the lymphocytes, by a dot blot technique. Data statistical analyses were performed by using the Student’s t-test (GraphPad Instat® 3.06 for Windows, GraphPad Software Inc., San Diego, CA, USA). Results. Statistically significant reductions of T and B lymphocyte proliferative response were observed, in TRT animals, at T1 (P<0.001 and P<0.05) and, to a limited extent, at T2. Serum IgM and IgG concentrations were found significantly higher than CTRL at T2 and T4, respectively. As regards the lymphocyte cytokines gene expression, an overall slight decrease of their expression was pointed out. Such a decrease was statistically significant (P<0.05), in the case of IL-8, at T2. Discussion and Conclusion. In the present study a significant decrease, at the early phase of the experiment, of the lymphoproliferative response was found. This was not surprising, as these blood sampling times were next to 17βE injections and it is known that oestrogens might decrease T and B cell lymphopoiesis (7). Such an effect was not observed later, in partial disagreement with previous studies which pointed out a possible immunosuppressive effect of both CLEN and DEX (8-9). However, it was recently reported that DEX, given at low doses to veal calves, reduced the lymphoproliferative response, whereas three different im administrations did not (3). Concerning the immunoglobulin data, the observed increase might be attributable to the administration of CLEN, as β-agonists have been shown to increase both serum IgG and IgM levels (10). The chosen cytokine gene expression levels were decreased pending all the experiment in TRT animals and, as a whole, confirm previously published data on each of the three drug used (11-13). The IS represents a target of many harmful chemicals and over the last ten years many efforts have been done to define tools able to predict either the level of exposure or the likely response to immunotoxicants in sentinel or target species. This study suggests that illicit drugs, given at low doses, might modulate the IS. Further studies are required to confirm and investigate these results