This paper describes a modification of the EC Four Plate Method based on microbial growth inhibition of Bacillus subtilis on agar medium at pH 6.0, 7.2 and 8.0 and Micrococcus luteus at pH 8.0 developed to cope with large numbers of samples. The method's performance was evaluated by determining the Minimum Inhibitory Concentrations of 66 commonly used drugs and determining the between-assay variation of antimicrobial control standards. The modified method proved particularly sensitive for beta-lactams, tetracyclines, quinolones, marcrolides and lincosamides and least sensitive for anticoccidials and nitrofurans. The pH 6.0 and 7.2 plates were more sensitive for 39 of the 66 antimicrobials (59%) whereas the two pH 8.0 plates (B. subtilis, M. luteus) were the most sensitive for 27 (41%). Muscle samples were taken from 1830 routine meat inspection investigations between 1994 and 1996. Of the 38 (2%) positive meat inspection carcasses, the following antimicrobials were confirmed above the MRL: penicillin G (10), oxytetracycline (16), sulphadimidine and sulphadiazine in combination (4) and chlortetracycline (1). The method as described is technically simple, cost effective, robust, suitable for large sample throughput and for frozen, thawed or fresh tissues. When all four plates are used the pattern of inhibition can reduce unnecessary confirmatory assays by indicating the antimicrobial group most likely to be present.
A study was carried out on 397 feedingstuffs and 11 premixes on which 161 (39.5%) were declared free of medication and 247 (60.5%) were medicated. These were subjected to analysis for the presence of antibiotics. Of the 247 medicated feeds 87 (35.2%) contained undeclared antimicrobials of which 59 (23.9%) were at a concentration sufficient to allow quantification by HPLC. Of the 161 unmedicated feeds 71 (44.1%) were shown to contain detectable antimicrobials of which 42 (26.1%) contained concentrations which could be quantified by HPLC. The most frequently identified contaminating antimicrobials were chlortetracycline (CTC) (15.2%), sulphonamides (6.9%), penicillin (3.4%) and ionophores (3.4%). Four samples (ionophores--3, sulphadimidine--1) contained therapeutic concentrations and one sample a supra-therapeutic concentration (monensin). The remainder were sub-therapeutic. All the contaminating concentrations of sulphadimidine detected were sufficient to cause violative tissue residues if fed to animals immediately prior to slaughter.
The administration of chloramphenicol (CAP) is banned in food animals in the European Union (EU). It is, therefore, important to have adequate screening methods to determine if residues of CAP and its major metabolite, chloramphenicol-glucuronide (CAP-Gluc), are present in samples taken for monitoring purposes. Six castrated male cattle were treated with a single intramuscular injection of 10 mg kg-1 CAP. Animals were sampled once daily for urine and were slaughtered at 3 and 6 d post-injection. Samples of bile, kidney, liver and diaphragmatic muscle were removed at slaughter. All matrices were analysed using the four plate test (FPT) bioassay, the Charm II radioimmunoassay and a Ridascreen CAP-Glucuronid competitive enzyme immunoassay (EIA). The FPT detected CAP residues in urine samples taken up to 2 d post-treatment. The Charm assay detected CAP in the urine for up to 4 d post-treatment. The EIA detected CAP throughout the 6 d sampling period. Samples of bile were positive by both the EIA and the Charm assay at day 3 and day 6. No zones of inhibition were obtained using the FPT in bile or diaphragm either with or without sample pre-treatment with beta-glucuronidase. However, the kidney and the liver from one animal killed at day 6 gave larger zones of inhibition after treatment with beta-glucuronidase, indicating the presence of CAP. The kidneys of all treated animals slaughtered at day 3 were positive by both the EIA and the Charm assay but none of the kidneys at day 6 tested positive by either method. Owing to technical difficulties, the Charm assay was not suitable for the analysis of liver. The EIA failed to detect CAP in the liver of any treated animal. It is concluded that urine appears to be the best matrix for screening purposes. The sensitivity of the FPT is inadequate for the determination of CAP residues were minimal withdrawal periods have been observed. The Charm assay and the EIA were suitable for the detection of both CAP and CAP-Gluc in tissues and body fluids for longer periods post-administration. The EIA was more sensitive for the determination of low concentrations of CAP and its metabolite.
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.
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.
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