Guy Degand

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.

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.

Monitoring Antibiotic Use and Residue in Freshwater Aquaculture for Domestic Use in Vietnam

Abstract Vietnam is an important producer of aquaculture products, and aquatic products are essential to the Vietnamese diet. However, Vietnam also has very little enforced regulation pertaining to antibiotic usage in domestic aquaculture, which raises concerns for antibiotic resistance in pathogenic bacteria. In this study, analysis was conducted on the presence of antibiotic residues in domestically sold fish and shrimp raised in freshwater farms in Vietnam, and an assessment of farmers’ knowledge of proper antibiotics usage was performed. The results indicated that a quarter of tested aquaculture products were antibiotic screening test positive, and there is a general lack of knowledge about the purpose and proper usage of antibiotics by aquaculture producers. Farmers’ decision-making processes about antimicrobial use are influenced by biased sources of information, such as drug manufacturers and sellers, and by financial incentives.

Determination of β-agonists in urine by an enzyme immunoassay based on the use of an anti-salbutamol antiserum

An enzyme immunoassay (EIA) method for β-agonists has been developed using an antiserum raised in rabbits by immunization against 3-O-salbutamol succinate coupled to bovine serum albumin. Horse radish peroxidase coupled to the same salbutamol derivative was selected as tracer. The dose of salbutamol which caused 50% binding inhibition was 16.8 pg per well and the limit of detection of the EIA directly performed on diluted urine was 0.14 ng ml−1 in urine (when the variability of blank values in samples from untreated animals was taken into account). Owing to the large cross reactivities of the antibodies with a number of β-agonists, the present assay is promising for the simultaneous determination of salbutamol, clenbuterol, mabuterol, terbutaline, cimaterol and probably also other β-agonists with a tert-butyl or isopropyl group at the extremity of the aliphatic side chain of the molecule.

Control of the illegal administration of natural steroid hormones in urine and tissues of veal calves and in plasma of bulls

In the context of the control of the illegal administration of natural steroid hormones in cattle husbandry, decision levels of sex steroid hormones were established, taking into account the effect of the treatment, for the cases of the urine of male and female veal calves treated with estradiol and/or testosterone-containing implants, and the plasma of bulls treated by an injection of an estradiol—testosterone cocktail. At each decision level, a score was assigned, that represents the percentage of treated animals detected when the decision limit is applied. Concerning the veal calves, a maximum decision level is proposed for the 17β-estradiol in urine of 1 ng ml−1 in both male and female veal calves, giving a score of 95%. A minimum decision level was set at 2 ng ml−1 for testosterone in urine of male veal calves, with a score of 95% (95% of the implanted male veal calves display a urinary testosterone level lower than 2 ng ml−1). For female veal calves, a maximum decision level was set at 0.45 ng ml−1 for the urinary testosterone concentration (score of 90%). For bulls, the 17β-estradiol concentration in plasma is a good criterion for detecting injected bulls: a maximum decision level was set at 40 pg ml−1, displaying a score ranging from 100 to 45%, 2 and 7 days after the injection, respectively.

Validation of a two-plate microbiological method for screening antibiotic residues in shrimp tissue

Microbiological inhibition screening tests could play an important role to detect residues of antibiotics in the different animal food products, but very few are available for the aquaculture products in general, and for shrimps in particular. A two-plate microbiological method to screen shrimp for residues of the most commonly used antibiotics has been developed and validated according to criteria derived from the European Commission Decision 2002/657/CE. Bacillus subtilis was used as a sensitive strain to target antibiotics. Culture conditions on Petri plates (pH of medium) were selected to enhance the capacity of antibiotic detection. Antibiotic residues were extracted from shrimps using acetonitrile/acetone (70/30, v/v) before application on Petri plates seeded with B. subtilis. The method was validated using spiked blank tissues as well as antibiotic treated shrimps with enrofloxacin and tetracycline, two antibiotics often found to be used in shrimp production. For tetracyclines and (fluoro)quinolones, the detection capability was below the maximum residue limit (MRL), while it was around the MRL for sulfonamides. The specificity of the microbiological screening was 100% in all cases while the sensitivity and accuracy was 100% in almost all cases. The capacity of the method to detect contaminated samples was confirmed on antibiotic treated shrimps, analyzed in parallel with a confirmatory method (Liquid Chromatography coupled to mass spectrometry (LC-MS)).

Determination of sulphamethazine in animal tissues by enzyme immunoassay

An enzyme immunoassay for sulphamethazine was developed using an antiserum raised in rabbits by immunization against a sulphamethazine diazo derivative coupled to bovine serum albumin. Horseradish peroxidase coupled to sulphamethazine by a carbodiimide method was selected as a tracer. The dose of sulphamethazine that caused 50% binding inhibition was 3.8 ng per well with a 2-h incubation time at 37°C and 1.2 ng per well with a 16-h incubation time at 4°C. The assay was evaluated by using control and sulphamethazine-fortified muscle and liver samples. These samples were treated by the matrix solid-phase dispersion method for the subsequent isolation of the drug. The method allowed a detection limit well below the tolerance limit (0.1 mg kg−1) generally applied for sulphamethazine.

Adaptation to multiday ozone exposure is associated with a sustained increase of bronchoalveolar uric acid.

The phenomenon of ozone tolerance is described, but the underlying mechanisms remain unknown. We tested whether adaptation to multiday ozone exposure was related to an upregulated pulmonary antioxidant defence. Six calves were exposed to 0.75 u ppm ozone, 12 u h u day m 1 for seven consecutive days. Pulmonary function tests and bronchoalveolar lavage (BAL) were performed before, after the first (D 1 ), third (D 3 ) and seventh (D 7 ) exposure. Differential cell count, total proteins, 8-epi-PGF 2 f , glutathione and uric acid were determined in BAL. Dynamic lung compliance and arterial oxygen tension were significantly decreased and lung oedema impaired pulmonary function on D 1 . By repeating ozone exposures, progressive functional adaptation occurred. Ozone induced a significant increase of BAL neutrophil percentage on D 1 . On D 3 and D 7 , neutrophil percentage was progressively decreased, but remained significantly elevated. BAL total proteins were significantly increased on D 1 and decreased progressively until D 7 . 8-Epi-PGF 2 f was significantly increased on D 1 and was returned to baseline on D 3 and D 7 , whilst glutathione significantly increased on D 3 and returned to baseline on D 7 . Uric acid was increased ten-fold on D 1 . On D 3 , uric acid was increased six-fold and was persistently elevated at D 7 . This study suggests that ozone adaptation of functional and inflammatory variables is accompanied with sustained BAL uric acid elevation.

Effect of storage and cooking on the fatty acid profile of omega-3 enriched eggs and pork meat marketed in Belgium

The fatty acids (FA) profile was determined in n-3 enriched (Columbus™) Belgian eggs and pork in order to evaluate to what extent the n-3 fatty acids, which are very sensitive to oxidation, are resistant to storage or cooking. In standard eggs or pork, no change of the fatty acid profile was observed after storage or cooking without culinary fat, as well as in Columbus™ eggs and pork after storage. Some cooking processes (eggs in custard and meat in oven) induced a slight significant loss of n-3 fatty acids in Columbus™ eggs or pork (11.1% in fat from eggs cooked in custard vs. 15.3% in raw Columbus™ eggs and 11.0% in fat from oven cooked meat vs. 11.6% in raw Columbus™ meat). As expected, when Columbus™ pork is cooked with culinary fat, its fatty acid profile is modified according to the nature of the fat used.