The Inhibitory Substance Test (IST), a microbiological growth inhibition test, is used for screening animal feedingstuffs for the presence of (contaminating) antimicrobial compounds. The effectiveness of the IST was established for 33 compounds that may be incorporated in feedingstuffs. Minimum detectable concentrations (MDCs) for standard solutions were established and compared with those obtained following solvent extraction of an antimicrobial-free compound feedingstuff spiked with each compound at 0-20 mg/kg. Of the 33 standard solutions examined, the test organism was not sensitive to 11 and the MDC for one was greater than its maximum inclusion rate in complete feedingstuffs. Following routine extraction (25% acetone-phosphate buffer) of feedingstuffs spiked with each of the 22 compounds to which the organism was sensitive, 10 were not detected, 15 were detectable at both minimum and maximum feed-inclusion rates and four were only detectable at their maximum feed-inclusion rates. Extraction with methanol (25%) had a deleterious effect with 12 compounds not detected, nine detectable at both minimum and maximum feed-inclusion rates and five detectable at their maximum feed-inclusion rates. Increasing acetone and methanol concentrations to 40 and 55% respectively resulted in larger inhibitory zones for antibiotic-free feedingstuff (25.3 + 2.43 mm vs 21.1 + 1.02 mm) compared with both 25% acetone (11.3 + 0.22 mm) and 25% methanol (11.2 + 0.22 mm), requiring the establishment of greater threshold zone diameters and negating any advantage in increasing the solvent concentration under these test conditions. It is concluded that the IST may be particularly useful for detection of a number of the zootechnical feed-additives recently banned in the EU, which, if used illegally, may be present at sufficiently high inclusion rates to facilitate detection. Further alteration of extraction conditions may improve the scope of the assay.
Poor recovery of the glycopeptide antibiotic avoparcin from certain animal feedstuffs, e.g., dairy feeds, has been experienced by this laboratory and has been reported elsewhere. It is postulated that this may be due to binding of avoparcin to specific carbohydrate-binding glycoproteins, known as lectins, which are naturally present in feedstuffs. The effect of sample pre-treatment with sugar solutions on the recovery of avoparcin was assessed. Samples (n = 17) of a bovine finishing feed containing 20 mg kg-1 avoparcin were pre-soaked with water (control) or one of two different sugar solutions (sucrose or methyl-aD-mannopyranoside) prior to extraction. Increases in avoparcin recoveries relative to the controls (1 .OOO) were (mean k standard error of the mean) 1.18 k 0.016 and 1.20 k 0.036 for methyl-aD-mannopyranoside and sucrose, respectively. In a second experiment, replicate samples (n = 4) of 30 different feedingstuffs were pre-soaked with methyl-aD-mannopyranoside and compared with the official extraction method. Pre-soaking elevated the mean avoparcin determinations for all the bovine and avian feedstuffs and for four out of seven porcine feedstuffs. The increased concentrations detected were significant for dairy (17.44 versus 10.31 mg kg-1; P < 0.001) and beef finishing feeds (23.12 versus 19.53 mg kg-1; P < 0.01). These results provide a possible solution to the low recoveries of avoparcin experienced in the assay of dairy feeds.
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