[Detection of processed animal protein: European experience and perspectives]. / Détection des protéines animales transformées: expérience et perspectives européennes.

European Commission Regulation (EC) No. 152/2009 imposes optical microscopy as the reference method for official controls to detect traces of animal protein in animal feed. Since 1 July 2004, the one-solvent technique has been the only authorised variant of optical microscopy. Its detection limit is 0.1% of meat-and-bone meal. Other techniques--using molecular biology (polymerase chain reaction, immunology), microscopy or near-infrared imaging--have been developed in the past ten years to supplement the official method, which has certain limitations. This paper compares and discusses the different techniques, highlighting the strengths of each technique in order to propose a feasible control scheme to improve the sensitivity and specificity of the technique for the detection of processed animal protein in livestock feed.

Imbalance between lipoxin A4 and leukotriene B4 in chronic mastitis-affected cows.

Persistent accumulation of inflammatory cells in the udder, with neutrophils being the predominant cell type, is a characteristic feature of chronic mastitis in dairy cows. Leukotriene (LT) B4 is a potent chemotactic agent, known to induce recruitment and accumulation of neutrophils in the bovine mammary gland. The LTB4-stimulated neutrophil functional responses are closely opposed by lipoxin (LX) A4, which promotes the resolution of inflammation. We thus hypothesized that the chronic inflammation of the udder could be associated with an unfavorable ratio between these two eicosanoids and that the persistence of neutrophil accumulation could be due to an increase in LTB4 synthesis and/or an impaired LXA4 production. In an attempt to verify this hypothesis, we first measured LXA4, LTB4, and their ratio in the milk of healthy and acute and chronic mastitis-affected quarters. Next, we studied the relationships between these variables and the degree of udder inflammation as assessed by somatic cell count measurement. The LTB4 concentration was low in healthy quarters, drastically increased in acute mastitis, and reached intermediate levels in chronic mastitis-affected quarters. However, whereas LXA4 concentration was highly increased in acute mastitis, healthy and chronic quarters had similarly low values. The LXA4:LTB4 ratio was thus significantly lower in chronic mastitis-affected cows. The LTB4 concentrations measured in chronic quarters were highly correlated to somatic cell count and to milk neutrophil and macrophage numbers. A weaker correlation was observed between LXA4 and these variables. For both eicosanoids, the highest correlation was observed with the number of neutrophils. These results show the existence of an LXA4:LTB4 imbalance in chronic mastitis-affected cows because of low LXA4 concentrations. Further studies are needed to determine whether administration of LX or stable analogs could have therapeutic potential in the control of chronic bovine mastitis.

Effect of nutritional antioxidant supplementation on systemic and pulmonary antioxidant status, airway inflammation and lung function in heaves-affected horses.

An oxidant/antioxidant imbalance in favour of oxidants has been identified as playing a decisive role in the pathogenesis of chronic inflammatory airway diseases. Nutritional antioxidant supplementation might reduce oxidative damage by enhancement of the antioxidant defence, thereby modulating inflammatory processes. In a placebo-controlled, blind study, it was tested whether a dietary antioxidant supplement administered for 4 weeks would improve lung function and reduce airway inflammation in heaves-affected horses. Eight horses in clinical remission of heaves were investigated at rest and after a standardised exercise test before and after treatment with an antioxidant supplement (consisting of a mixture of natural antioxidants including vitamins E and C and selenium from a variety of sources) or placebo (oatfeed pellets without additive). Pulmonary function and exercise tolerance were monitored; systemic and pulmonary lining fluid uric acid, glutathione and 8-epi-PGF(2alpha) were analysed, and bronchoalveolar lavage (BAL) cytology and inflammatory scoring of the airways were performed. The antioxidant treatment significantly improved exercise tolerance and significantly reduced endoscopic inflammatory score. Plasma uric acid concentrations were significantly reduced, suggesting downregulation of the xanthine-dehydrogenase and xanthine-oxydase pathway. Haemolysate glutathione showed a nonsignificant trend to increase, while plasma 8-epi-PGF(2alpha) remained unchanged. Pulmonary markers and BAL cytology were not significantly affected by antioxidant supplementation. The present study suggests that the antioxidant supplement tested modulated oxidant/antioxidant balance and airway inflammation of heaves-affected horses.

Effect of chronic airway inflammation and exercise on pulmonary and systemic antioxidant status of healthy and heaves-affected horses.

In heaves-affected horses the relation between oxidant status, airway inflammation (AI) and pulmonary function (PF) is unknown. The oxidant status of blood and pulmonary epithelial lining fluid (PELF) of healthy (H, n = 6) and heaves-affected horses in clinical remission (REM, n = 6) and in crisis (CR, n = 7) was assessed at rest, during and after standardised exercise test by measurement of reduced and oxidised glutathione, glutathione redox ratio [GRR%]; uric acid and 8-epi-PGF2alpha. Oxidant status was related to PF parameters (mechanics of breathing and arterial blood gas tension) and Al parameters (bronchoalveolar lavage [BAL] neutrophil % and AI score). Haemolysate glutathione was significantly different between groups and was correlated with PF and AI parameters; GRR in PELF was increased during CR and was correlated with PF and AI parameters. Exercise induced an increase of plasma uric acid that was significantly higher both in REM and CR. PELF 8-epi-PGF2alpha was significantly increased in CR and correlated with PF and AI parameters. These results suggest that oxidative stress occurring in heaves is correlated with PF and AI and may be locally assessed by PELF glutathione status, uric acid and 8-epi-PGF2alpha. Systemic repercussions are reflected by assay of GSH in resting horses and by uric acid in exercising horses.

Control of the illegal administration of natural steroid hormones in the plasma of bulls and heifers.

In the context of the control of the illegal administration of natural steroid hormones in cattle husbandry, an attempt was made to establish the decision levels for sex steroid hormones in the plasma of adult cattle, taking into account the effect of the treatment. Bulls and heifers were treated with two injections, at a two week interval, of an estradiol-testosterone cocktail. Steroid hormone and biochemical precursor concentrations were measured in plasma samples by using specific radioimmunoassays, before and after the treatment. When the treatment significantly (p < 0.05) modified a hormone concentration, a decision level was established for that hormone concentration. At each decision level, a score was assigned that represented the percentage of treated animals detected when the decision limit was applied. For heifers, 17 beta-estradiol and testosterone concentrations in plasma, which increased after the treatment, are the best criteria to use to detect treated animals, with decision limits of 20 pg ml-1 and 125 pg ml-1, respectively. In the instance of bulls, both testosterone and steroid biochemical precursor concentrations decreased in the plasma after the treatment. We proposed decision limits of 1500 pg ml-1 and 28 pg ml-1 for testosterone and androstenedione concentrations, respectively, the bulls displaying concentrations below these limits being positive. We observed that the repetition of the injection increased the score of the decision limit. The scores for testosterone are 70%, 14d after the first injection and 100% 14 d after the second injection, and for androstenedione, these scores are 60 and 100%, respectively.

[Polyarthritis patients with and without detected rheumatoid factor: a comparison of the psychological personality]. / Polyarthritis-Patienten mit und ohne Rheumafaktor-Nachweis: Ein persönlichkeitspsychologischer Vergleich.

The study examined again the question whether the presence of rheumatoid factor in rheumatoid arthritis (rh.A.) patients has a bearing on their personality scores. For this purpose 20 seropositive rh.A. patients were compared by means of the FPI-R (Freiburg Personality Inventory) with 20 rh.A. patients in whom the rheumatoid factor was absent. Groups were matched for sex and age; severity and duration of disease were taken into account as covariates. The covariates showed significant correlations with some personality variables. Diagnostic subgroups, however, did not differ in any of the FPI scales.

Anabolics: the situation in Belgium.

The illegal use of anabolics in 1989-1990 and its control in Belgium are reviewed. After a short presentation of the Belgian legislation and EEC directives, a recent hormone scandal published by a consumer magazine is related and the composition of 17 hormone cocktails found on the black market is given. The strategy of control involves screening of the main artificial anabolics in urine by radioimmunoassay (RIA) and confirmation by thin-layer chromatography (TLC). Samples of fat and injection sites are also examined by TLC. In the future, RIA will probably be replaced by enzyme immunoassay and TLC by mass spectrometry.

Development of a specific radioimmunoassay for the detection of clenbuterol residues in treated cattle.

A radioimmunoassay for clenbuterol detection in cattle has been validated and used to monitor treated cattle. The tracer used was 4-amino-3,5-dichloro-alpha(tert-butylamino-methyl) benzyl alcohol (benzyl-3H)(clenbuterol) prepared by catalytic tritiation with tritium gas of 4-amino-3,5-dibromo-alpha-(tert-butylamino)-acetophenone, followed by chlorination at positions 3 and 5 in the aromatic ring. The rabbit antiserum was raised against a diazotized clenbuterol/human serum albumin conjugate. The assay described was sensitive (7.8 pg/tube) and reproducible. The intra- and inter-assay variability, which was assessed by measuring known quantities of clenbuterol in plasma, urine and faeces, was satisfactory for RIA. When this assay was used to monitor treated cattle the concentrations of clenbuterol in plasma, urine and faeces were directly related to the administered dose. The absorption and elimination of clenbuterol in cattle was rapid. Data obtained were consistent with results obtained in other species where a rapid clearance rate was also demonstrated.

Enzyme immunoassay screening procedure for the synthetic anabolic estrogens and androgens diethylstilbestrol, nortestosterone, methyltestosterone and trenbolone in bovine urine.

Immunoassays are often used for the screening of anabolic residues in edible tissues and excreta (urine, faeces) from inspected animals. Radioimmunoassays have been used for ten years for the determination in biological samples of the main natural and synthetic anabolic estrogens and androgens. In order to simplify the sample preparation and analysis and to reduce the cost, competitive enzyme immunoassays (EIA) were developed for the main synthetic anabolics used illegally in livestock fattening. EIA are based on a competition between the analyte (hormone or metabolite) and the enzyme-labelled hormone for binding to specific antibodies immobilized in wells of a microtitration plate. Two enzymes were evaluated: horseradish peroxidase (HRP) and Bacillus licheniformis beta-lactamase (BLL) using hydrogen peroxide-o-phenylenediamine or benzylpenicillin-starch-iodine as substrates, respectively. The same derivative was used for chemical coupling of the hormone to enzyme (tracer preparation) and to bovine serum albumin to produce specific antibodies in rabbits. Hormone doses that inhibited 50% of the tracer (HRP-hormone) binding to antibody (ID50) were 18, 8, 6 and 11 pg per well for diethylstilbestrol, nortestosterone, methyltestosterone and trenbolone, respectively. These values were lower than those observed in RIA. The reproducibility and accuracy of EIA in urine analysis were similar to those of RIA. Very small amounts of urine were needed (2.5 microliters). This simple method may require less than 2 h. With the BLL-hormone tracer, the enzymatic activity remaining in the wells and hence the hormone content of the sample could be estimated with the naked eye using benzylpenicillin-starch-iodine as substrate.