A possible correlation between the blood leukocyte formula and the use of glucocorticoids as growth promoters in beef cattle.

Clinical analyses designed to set welfare parameters were performed on blood drawn from the caudal vein of 14 groups of cattle (young bulls and heifers) (n = 10) from 480 to 550 kg b.w., each group representative of a different farm. The leukocyte formula exhibited a lymphocytopenia in four groups compared with the values from a control group (n = 50). This finding was related to the possible illicit use of corticoids as growth promoters in meat production. The individual plasmas tested negative by two different ELISA kits for corticosteroids, but chemical analyses by LC-MS/APCI (detection limit 0.5 ng/ml) on the pooled plasma of each of the 14 groups revealed the presence of beclomethasone and fluocinolone acetonide in 3 of the 4 suspect farms. These corticosteroids are not always efficiently screened by commercially available immunoassays. The epidemiological reliability of blood analysis as a screening test for such drugs is discussed in the light of the need for quality certification of the whole meat production processes 'from farm to fork', and for enhanced animal welfare.

Identification of main corticosteroids as illegal feed additives in milk replacers by liquid chromatography-atmospheric pressure chemical ionisation mass spectrometry.

Corticosteroids were proposed as growth promoting agents to improve commercial quality of meat. We developed a liquid chromatography-atmospheric pressure chemical ionisation mass spectrometry (LC-APCI-MS) method able to identify the presence in milk replacers, when given by mouth, of dexamethasone, betamethasone, flumethasone, triamcinolone, predinisotone, prednisolone, methylprednisolone, fludrocortisone and beclomethasone, at levels in the range of 20-100 ppb. C18 solid-phase extraction, LC-RP C8 column separation, data acquisition (positive ions) in the scan range m/z 200-550 allowed us to differentiate and identify compounds by protonated molecules, their methanolic adducts and fragmentation patterns.

Synovial fluid as a matrix of selection in the detection of beta-adrenergic agonist drugs in carcases and fresh meat.

Previous papers have supported the hypothesis that beta-agonist drugs could accumulate in those tissues with a high content of mucopolysaccharides. Based on our preliminary findings, between October and December 1993 we randomly sampled 534 samples of synovial fluids drawn from the knee joint of fresh carcasses. After a preliminary extraction able to break down the water domains of mucopolysaccharides and the interactions between the matrix and the drugs, samples were concentrated on diatomaceous earth and then screened on two different ELISA plates. We confirmed the structure of suspected fluids by GC-MS (HFBA derivatization), according to EC criteria. Of the 57 samples screened as positive (10.6% of the total), 51 (9.5%) were fully confirmed, while for six it was not possible to identify the drug. The results suggest that the analysis of synovial fluids is an adequate tool to monitor the misuse of beta-adrenergic drugs in animal production, especially when target organs such as liver and kidney are not available for sampling.

Anatoxin-A and a previously unknown toxin in Anabaena planctonica from blooms found in Lake Mulargia (Italy).

Algal extracts of Anabaena planctonica from Lake Mulargia in Italy were tested for toxins by mouse bioassay, the Microtox system, and GC-MS and HPLC chromatography. Anatoxin-a was identified by GC-MS after derivatization with pentafluorobenzyl bromide. Hepatotoxins were also present and are perhaps related to the microcystins present in other species of Anabaena.

Microcystin-like toxins in different freshwater species of Oscillatoria.

In January and September of 1989 and March 1990 blooms of Oscillatoria rubescens, Oscillatoria tenuis and Oscillatoria mougetii were found in Lake Simbirizzi and Lake Flumendosa in Sardinia, and in Lake San Puoto in the Lazio region of Italy. By using different extraction methods and HPLC analysis, two microcystin-like toxins (RR-like and YR-like), similar to some of the toxic compounds produced by the Cyanophycea Microcystis aeruginosa, were detected in these blooms.

Presence of saxitoxin in toxic extracts from Gonyaulax polyedra.

A "red tide" bloom of Gonyaulax polyedra occurred in Italy in Autumn, 1988. Algal concentrated extracts and undiluted water samples from the bloom were tested both with the Microtox system and a mouse bioassay, revealing the presence of paralytic shellfish poison-like neurotoxins. Saxitoxin levels evaluated on the basis of toxicological and instrumental analysis showed discrepancies. Other toxins could be present in addition to paralytic shellfish poison.

Characterization and some properties of the venom gland extract of a theridiid spider (Steatoda paykulliana) frequently mistaken for black widow spider (Latrodectus tredecimguttatus).

The simplified purification protocol established for the isolation of alpha-latrotoxin from the venom of the spider Latrodectus tredecimguttatus, has been employed for the purification of toxic components present in the venom of the spider Steatoda paykulliana. The venom of this spider, frequently mistaken for L. tredecimguttatus, is by tradition considered to cause an envenomation potentially dangerous to man. The venom of S. paykulliana has little toxic effect on guinea-pigs but is extremely toxic to houseflies (Musca domestica). No proteolytic activity was detectable. Interaction of microgram/ml amounts of the venom extract with artificial lipid membranes produces an increase of membrane conductance through the formation of stable ion-permeable channels modulated by the direction and size of the electric potential differences across the membrane. Higher concentrations of this venom are able to stimulate the release of transmitters from neurosecretory cells in a fashion reminiscent of black widow spider venom. Antibodies against the whole L. tredecimguttatus venom gave a few positive cross-reactions in the immunodiffusion test with S. paykulliana venom gland extract indicating the presence of common molecular sequences in the two venoms. Polyclonal antibodies against alpha-latrotoxin did not cross-react in the immunodiffusion test with S. paykulliana venom extracts, nor in the immunofluorescence assay with its cephalothorax sections, thus suggesting that the venom glands do not contain alpha-latrotoxin. A partial characterization of S. paykulliana venom has been performed and a high molecular weight protein toxic to houseflies has been partially purified.