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In the combat against bacterial resistance, there is a clear need to check the use of antibiotics in animal husbandry, including poultry breeding. The use of chicken feathers as a tool for the detection of use of antibiotics was investigated. An extraction method for the analysis of oxytetracycline (OTC) from feathers was developed and was tested by using incurred feathers obtained from a controlled animal treatment study. The use of McIlvain-ethylenediaminetetraacetic acid buffer only in combination with acetone gave the highest extraction yield, indicating the need of an organic solvent for feather extraction. By using the developed method, it was found that after a withdrawal time, the OTC concentration in feathers is in the mg kg⁻¹ range, far higher than that in muscle and liver tissue. Based on the analysis of individual segments of feathers from OTC-treated chicken, evidence was found supporting the hypothesis of secretion of antibiotics through the uropygial gland and external spread over feathers by grooming behaviour. It was also found that part of the administered OTC is built into the feather rachis. Finally, we provide the first evidence that the analysis of individual segments of the rachis can be used as a tool to discriminate among different treatment strategies, for example, therapeutic versus subtherapeutic. As a result, we concluded that the analysis of feathers is an extremely valuable tool in residue analysis of antibiotics.

len. Validation and application of a robust yeast estrogen bioassay for the screening of estrogenic activity in animal feed. Food Additives and Contaminants, 2006, 23 (06) . All of these blank and low estrogen spiked feed samples fulfilled the CCα and CCβ criterions, meaning that all 20 blank feed samples gave a signal below the determined decision limit CCα and were thus classified as compliant and at least 19 out of the 20 spiked samples gave a signal above this CCα (β=5%) and were thus classified as suspect. The method was specific and estrogens in feed were stable for up to 98 days. In this study we also present long-term performance data and several examples of estrogens found in the routine screening of animal feed.

A number of metabolites of 17b-estradiol were tested for their estrogenic activity using the ER-CA-LUX assay based on the increased expression of luciferase in exposed T47D breast cancer cells. E 2 b and estrone showed similar potencies in the test, whereas E 2 a was 100 times less active. Incubation of cells with estrone (0.35 mM) resulted in the formation of E 2 b, whereas the reverse reaction was observed for E 2 b. The resulting equilibrium may explain the similar estrogenic potency of estrone in the test. The synthetic 17-hydroxy benzoate ester of E 2 b was 3 times less active than the parent compound. The 17-hydroxy palmitate and oleate esters of E 2 b, were respectively 25 and 200 times less active than the parent compound. The 2-hydroxy metabolites of E 2 b and estrone showed a 5,000 to 10,000 fold lower activity. The 4-hydroxy metabolites were more potent than the 2-hydroxy metabolites, showing only a 20-200 times lower activity. The 2-and 4-methoxyesters of estrone were 700 times less active. It is concluded that the estrogenic potency of metabolites formed in cattle after treatment with E 2 b, like estrone, E 2 a and especially the esters of E 2 b, may be significant with respect to the potential risk of the use of estradiol for growth promotion in domestic animals in certain countries.Key words: Estrogenicity; ER-CALUX; catecholestrogens; estradiol; estradiol-esters. L. A. P. Hoogenboom, RIKILT, Bornsesteeg 45, 6708PD Wageningen, The Netherlands, e-mail: L.A.P. Hoogenboom/rikilt.wag-ur.nl The use of 17b-estradiol for growth-promoting purposes in cattle may result in the increased formation of residues of not only the parent compound but also its metabolites. In order to investigate the potential risk for the consumer, it is essential to obtain information on the identity, levels and biological properties of these compounds. In cattle the major metabolites of E 2 b are estrone, the 17a-congener and their glucuronide conjugates (1-3). Previous studies showed increased levels of the parent compound, and the two major bovine metabolites,

Recent studies indicate that next to antibiotic resistance, bacteria are able to subsist on antibiotics as a carbon source. Here we evaluated the potential of gut bacteria from healthy human volunteers and zoo animals to subsist on antibiotics. Nine gut isolates of Escherichia coli and Cellulosimicrobium sp. displayed increases in colony forming units (CFU) during incubations in minimal medium with only antibiotics added, i.e., the antibiotic subsistence phenotype. Furthermore, laboratory strains of E. coli and Pseudomonas putida equipped with the aminoglycoside 3′ phosphotransferase II gene also displayed the subsistence phenotype on aminoglycosides. In order to address which endogenous genes could be involved in these subsistence phenotypes, the broad-range glycosyl-hydrolase inhibiting iminosugar deoxynojirimycin (DNJ) was used. Addition of DNJ to minimal medium containing glucose showed initial growth retardation of resistant E. coli, which was rapidly recovered to normal growth. In contrast, addition of DNJ to minimal medium containing kanamycin arrested resistant E. coli growth, suggesting that glycosyl-hydrolases were involved in the subsistence phenotype. However, antibiotic degradation experiments showed no reduction in kanamycin, even though the number of CFUs increased. Although antibiotic subsistence phenotypes are readily observed in bacterial species, and are even found in susceptible laboratory strains carrying standard resistance genes, we conclude there is a discrepancy between the observed antibiotic subsistence phenotype and actual antibiotic degradation. Based on these results we can hypothesize that aminoglycoside modifying enzymes might first inactivate the antibiotic (i.e., by acetylation of amino groups, modification of hydroxyl groups by adenylation and phosphorylation respectively), before the subsequent action of catabolic enzymes. Even though we do not dispute that antibiotics could be used as a single carbon source, our observations show that antibiotic subsistence should be carefully examined with precise degradation studies, and that its mechanistic basis remains inconclusive.

A number of metabolites of 17beta-estradiol were tested for their estrogenic activity using the ER-CA-LUX assay based on the increased expression of luciferase in exposed T47D breast cancer cells. E2beta and estrone showed similar potencies in the test, whereas E2alpha was 100 times less active. Incubation of cells with estrone (0.35 microM) resulted in the formation of E2beta, whereas the reverse reaction was observed for E2beta. The resulting equilibrium may explain the similar estrogenic potency of estrone in the test. The synthetic 17-hydroxy benzoate ester of E2beta was 3 times less active than the parent compound. The 17-hydroxy palmitate and oleate esters of E2beta, were respectively 25 and 200 times less active than the parent compound. The 2-hydroxy metabolites of E2beta and estrone showed a 5,000 to 10,000 fold lower activity. The 4-hydroxy metabolites were more potent than the 2-hydroxy metabolites, showing only a 20-200 times lower activity. The 2- and 4-methoxyesters of estrone were 700 times less active. It is concluded that the estrogenic potency of metabolites formed in cattle after treatment with E2beta, like estrone, E2alpha and especially the esters of E2beta, may be significant with respect to the potential risk of the use of estradiol for growth promotion in domestic animals in certain countries.

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