Microbial screening methods for detection of antibiotic residues in slaughter animals.
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ABSTRACT:
SUBMITTER: Pikkemaat MG
PROVIDER: S-EPMC2758187 | biostudies-other | 2009 Oct
REPOSITORIES: biostudies-other
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Project description:Objective:To analyze the presence of selective antibiotic residues (oxytetracycline, amoxicillin, and ciprofloxacin) in milk during the antibiotic treatment course, and to evaluate the thermal effect on antibiotics residual status in milk of antibiotic-treated cows. Materials and Methods:The raw fresh milk was collected from 18 lactating cows before antibiotics treatment, which were brought to the veterinary hospital and suffered from either mastitis, foot and mouth disease, fever, local wound, or non-specific diarrhea, and so on. Out of the 18 lactating cows, six were treated with oxytetracycline, six were treated with amoxicillin, and six were treated with ciprofloxacin parenterally. Milk samples were also collected at 2nd day during treatment and final collection was done after maintaining the withdrawal period. Since milk is heated before consumption, it was boiled at 100°C for 20 min to evaluate the thermal effect on antibiotics residual status. Thin-layer chromatography was done for screening of antibiotics residue before and after boiling of the milk. Results:At day 0 (before antibiotic treatment), no antibiotics (oxytetracycline, ciprofloxacin and amoxicillin) residue was detected in raw milk of antibiotic treated cows. In contrast, on day 2 (during antibiotic treatment), 100% raw milk samples showed positive for antibiotics residue. After boiling, all milk samples showed positive for such specific antibiotics residue. On the other hand, no antibiotics residues were detected on day 9, which indicates the completion of the withdrawal period of the respective antibiotic. The intensities of bands for antibiotic on thin-layer chromatography plate of antibiotic residues in milk samples (oxytetracycline, amoxicillin, and ciprofloxacin) expressed that the respective antibiotic residual status was higher in the boiled milk compare to the raw milk. Conclusion:Proper maintenance of withdrawal period after antibiotic treatment would minimize the risk of antibiotic residues in milk, and boiling does not change these specific antibiotics residual status in milk. Therefore, awareness regarding the proper maintenance of withdrawal period after antibiotic treatment in lactating cows is one of the best strategies that may positively reduce the risk of antimicrobial drugs residue in milk.
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Project description:In the present study, antibiotic residues were detected in milk samples collected from the dairy herds located in Karnataka, India, by microbiological assay. Subsequently, the detected antibiotics were identified as azithromycin and tetracycline, by high-performance liquid chromatography, further both the antibiotics detected in the cow milk samples were found to be at high concentration (9708.7 and 5460 ?g kg-1, respectively). We then investigated the effects of temperature and pH on the stabilities of azithromycin and tetracycline to determine the degradation rate constant k using first-order kinetic equation. Results indicated that significant reduction in stability and antibacterial activity of azithromycin solution when subjected to 70 and 100°C for 24 h. While stability of tetracycline was significantly reduced when subjected to 70 and 100°C for 24 h. However no significant reduction in antibacterial activity of tetracycline was observed at respective temperatures when compared with that of control. In addition, the stabilities of azithromycin and tetracycline were found to be decreased in acidic pH 4-5. The results of the present study revealed the high risk of contamination of milk sample with veterinary antibiotics and also demonstrated the effect of temperature and pH on stability of antibiotics. Therefore the study suggest that the qualitative and quantitative screening of milk for the presence of antibiotics need to be strictly performed to ensure safe drinking milk for consumers.
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Project description:BackgroundQuantitative polymerase chain reaction (qPCR) assays used in pathogen detection require rigorous methods development including characterizing DNA extraction products. A DNA extract characterization process is demonstrated using DNA extracted from five different cells types (two Gram-negatives: Escherichia coli, and Burkholderia thailandensis, spores and vegetative cells from the Gram-positive Bacillus cereus, and yeast Saccharomyces cerevisiae) with six different methods.ResultsDNA extract quantity (concentration and extraction efficiency) and quality (purity and intactness) varied by cell type and extraction method enabling the demonstration of different DNA characterization methods. DNA purity was measured using UV spectroscopy, where the A260/A280 and A260/A230 ratios are indicators of different contaminants. Reproducibility of UV spectroscopy measurements decreased for DNA concentrations less than 17.5 ng/?L. Forty-seven extracts had concentrations greater than 17.5 ng/?L, 25 had A260/A280 above 2.0, and 28 had A260/A230 ratios below 1.8 indicating RNA and polysaccharide contamination respectively. Based on a qPCR inhibition assay the contaminants did not inhibit PCR. Extract intactness was evaluated using microfluidic gel electrophoresis. Thirty-five samples had concentrations above the limit of quantification (LOQ, roughly 11 ng/ ?L), 93.5% of the DNA was larger than 1kb and 1% was smaller than 300 bp. Extract concentrations ranged from 1502.2 ng/?L to below the LOQ when UV spectroscopy, fluorometry, and qPCR were used. LOQ for UV spectroscopic and fluorometric measurements were 3.5 ng/?L and 0.25 ng/?L respectively. The qPCR LOQ varied by cell type (5.72?×?10-3 ng/?L for E. coli, 2.66?×?10-3 ng/?L, for B. cereus, 3.78?×?10-3 ng/?L for B. thailandensis, and 7.67?×?10-4 ng/?L for S. cerevisiae). A number of samples were below the UV spectroscopy (n?=?27), flurometry (n?=?15), and qPCR (n?=?3) LOQ.ConclusionThe presented DNA extract characterization process provides measures of DNA quantity and quality applicable to microbial detection methods development and validation studies. Evaluating DNA quality and quantity results in a better understanding of process LOD and contributing factors to suboptimal assay performance. The samples used demonstrated the use of different DNA characterization methods presented but did not encompass the full range of DNA extract characteristics.
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