Solvent Front Position Extraction is a novel technique developed for effective sample preparation of biological samples containing coccidiostats prior to LC-MS/MS. In this study the technique was ...used for isolation and determination of seven coccidiostats, from both main groups being: ionophores and chemical coccidiostats. Its effectiveness was evaluated by comparing with other sample preparation procedures, used in European routine laboratories. Results obtained by Solvent Front Position Extraction were very satisfactory (linearity R
≥ 0.971, recovery 90.1-111.1%, RSDr: 8.7-16.6%, RSD
: 9.0-17.7%) and fulfilled requirements described in Commission Regulation (EU) 2021/808 of 22 March 2021, which showed great potential of the technique in sample preparation of coccidiostats in poultry feed.
A multianalyte method was developed to identify and quantitate 26 mycotoxins simultaneously in maize silage by means of ultrahigh-performance liquid chromatography–tandem mass spectrometry ...(UHPLC-MS/MS). The extraction and cleanup procedure consists of two extraction steps followed by purification on a Waters Oasis HLB column. The method developed was validated with the requirements of Commission Decision 2002/657/EC taken into account. The limit of detection and quantitation ranges were 5–348 and 11–695 ng/g, respectively. Apparent recovery varied between 61 and 116%, whereas repeatability and reproducibility were within the ranges of 3–45 and 5–49%, respectively. The method developed was successfully applied for maize silage samples taken at the cutting surface and 1 m behind that surface. Mainly Fusarium toxins (beauvericin, deoxynivalenol, enniatins, fumonisins, fusaric acid, and zearalenone) were detected, but postharvest toxins such as mycophenolic acid and roquefortine C were identified as well.
Fertilization with animal manure is one of the main routes responsible for the introduction of antibiotic residues, antibiotic resistance genes, and zoonotic bacteria into the environment. The aim of ...this study was to assess the effect of the use of pig (swine) manure as a fertilizer on the presence and fate of six antibiotic residues, nine antibiotic resistance genes, and bacteria (zoonotic bacteria
Salmonella
spp. and
Campylobacter
spp. and
E. coli
as indicator for Gram-negative bacterial species of the microbiota of livestock) on five fields. To the best of our knowledge, the present study is the first to assess a multitude of antibiotic residues and resistance to several classes of antibiotics in pig manure and in fertilized soil over time in a region with an intensive pig industry (Flanders, Belgium). The fields were sampled at five consecutive time points, starting before fertilization up to harvest. Low concentrations of antibiotic residues could be observed in the soils until harvest. The antibiotic resistance genes studied were already present at background levels in the soil environment prior to fertilization, but after fertilization with pig manure, an increase in relative abundance was observed for most of them, followed by a decline back to background levels by harvest-time on all of the fields studied. No apparent differences regarding the presence of antibiotic resistance genes in soils were observed between those fertilized with manure that either contained antibiotic residues or not. With regard to dissemination of resistance, the results presented in this study confirm that fertilization with animal manure directly adds resistance genes to the soil. In addition, it shows that this direct mechanism may be more important than possible selective pressure in soil-dwelling bacteria exerted by antibiotic residues present in the manure. These results also indicate that zoonotic bacteria detected in the manure could be detected in the soil environment directly after fertilization, but not after 1 month. In conclusion, although some antibiotic residues may be present in both manure and soil at concentrations to exert selective pressure, it seems that antibiotic resistance is mostly introduced directly to soil through fertilization with animal manure.
This paper describes a method to detect and quantitate the endogenous plant hormones (±)-2-cis-4-trans-abscisic acid, (−)-jasmonic acid, and salicylic acid by means of ultrahigh-performance liquid ...chromatography–tandem mass spectrometry (UHPLC–MS/MS) in hybrid rose leaf matrices. Deuterium-labeled 2H6 (+)-2-cis-4-trans-abscisic acid, 2H6 (±)-jasmonic acid, and 2H4-salicylic acid were used as internal standards. Rose samples (10 mg) were extracted with methanol/water/acetic acid (10:89:1) and subsequently purified on an Oasis MCX 1 cm3 Vac SPE cartridge. Performance characteristics were validated according to Commission Decision 2002/657/EC. Recovery, repeatability, and within-laboratory reproducibility were acceptable for all phytohormones tested at three different concentrations. The decision limit and detection capability for (±)-2-cis-4-trans-abscisic acid, (−)-jasmonic acid, and salicylic acid were 0.0075 and 0.015 μg/g, 0.00015 and 0.00030 μg/g, and 0.0089 and 0.018 μg/g, respectively. Matrix effects (signal suppression or enhancement) appeared to be high for all substances considered, implying the need for quantitation based on matrix-matched calibration curves.
Pig manure may contain antibiotic residues, antibiotic-resistant bacteria or pathogens, which may reach the environment upon fertilization. During this study, 69 antibiotic residues belonging to 12 ...classes were quantified in 89 pig slurry samples. These samples were also studied for the presence of
and for
resistant to meropenem, colistin, ciprofloxacin, or cefotaxim. The obtained isolates were further tested for antibacterial susceptibility. No antibiotic residues were detected in four samples, whereas in the other samples, up to 12 antibiotics were found. The most frequently detected antibiotic residues were doxycycline, sulfadiazine, and lincomycin. Doxycycline was found in the highest concentration with a mean of 1476 µg/kg manure (range: 18-13632 µg/kg). Tylosin and oxytetracycline were found with mean concentrations of 784 µg/kg (range: 17-5599 µg/kg) and 482 µg/kg (range: 11-3865 µg/kg), respectively. Lincomycin, had a mean concentration of 177 µg/kg manure (range: 9-3154 µg/kg). All other 18 antibiotic residues were found with mean concentrations of less than 100 µg/kg manure. Fifty-one slurry samples harbored
; 35% of the
isolates were sensitive to a panel of 14 antibiotics, whereas the other 65% were resistant up to five antibiotics. For
, 52 manure samples contained
isolates which were resistant to ciprofloxacin and 22 resistant to cefotaxime. All ciprofloxacin and cefotaxime-resistant isolates were multi-resistant, with resistance up to nine and eight antibiotics, respectively. This research indicates that pig slurry used for fertilization often contains antibiotic residues and antibiotic-resistant bacteria, including pathogens.
A molecularly imprinted polymer (MIP) was designed for benzylpenicillin via suspension polymerization. The specific absorption of benzylpenicillin to the MIP, applied in a molecularly imprinted ...solid-phase extraction (MISPE), was compared to the nonspecific binding using a NIP (nonimprinted polymer without a target molecule) in a non-molecularly imprinted solid-phase extraction. This validation was performed successfully in acetonitrile solutions and milk extracts spiked with benzylpenicillin. Significant differences in absorption were observed. In acetonitrile, the recoveries using MISPE (90–95%) were a fraction higher than those in milk extracts (70–80%). The validation revealed the limit of detection and the limit of quantitation for the MISPE application in milk samples to be 0.51 and 1.02 μg/kg, respectively. In addition, comparing the results of the analysis of positive milk samples using MISPE with those using a classic sample preparation step showed a Pearson correlation of 0.989. Finally, cross reactivity tests using other antibiotics showed a certain cross reactivity, but non-β-lactams were barely bound.
Antibiotic resistant bacteria and antibiotic residues can enter the environment when using animal manure as fertilizer. Twenty-five mixed beef cattle farmyard manure samples and 9 mixed fattening ...calf slurry samples from different farms across Belgium were investigated for the presence of 69 antibiotic residues, antibiotic resistant
and
spp. Doxycycline, oxytetracycline, ciprofloxacin, enrofloxacin, flumequine and lincomycin were detected in all fattening calf slurry samples with mean concentrations of 2776, 4078, 48, 31, 536 and 36 µg/kg manure, respectively. Sulfadiazine was detected at a mean concentration of 10,895 µg/kg. Further, antibiotic residues were found in only 4 of the 25 beef cattle farmyard manure samples. Oxytetracycline was detected twice below 500 µg/kg. Paromomycin, ciprofloxacin and enrofloxacin were detected in a concentration below 100 µg/kg. Of
isolates, 88% and 23% from fattening calf slurry and beef cattle farmyard manure, respectively, were resistant to at least one of the antibiotics tested. Multi-drug resistance was observed at a maximum of 10 and 7 antibiotics, respectively. The occurrence of antibiotic resistant
and antibiotic residues is shown to be higher in fattening calf slurry than in beef cattle farmyard manure used for agricultural field fertilization.
When crops are cultivated on fields fertilized with animal manure, the risk exists that plants may take up antibiotic residues and may be exposed to antibiotic resistance genes and antibiotic ...resistant bacteria. During cultivation in a greenhouse pot experiment, leek (Allium porrum) was fertilized with either pig slurry or mineral fertilizer and exposed to either no antibiotics, doxycycline (10,000 μg/kg manure), sulfadiazine (1000 μg/kg manure), or lincomycin (1000 μg/kg manure). At harvest, 4.5 months later, lincomycin, sulfadiazine or doxycycline were not detected in any of the leek samples nor in their corresponding soil samples. Further, antimicrobial susceptibility testing was performed on 181 Bacillus cereus group isolates and 52 Pseudomonas aeruginosa isolates from the grown leek. For the B. cereus group isolates, only a small shift in MIC50 for lincomycin was observed among isolates from the lincomycin and control treatment. For P. aeruginosa, only in the setup with doxycycline treatment a higher MIC50 for doxycycline was observed compared to the control, specifically the isolates selected from growth media supplemented with 8 mg/L doxycycline. Nine antibiotic resistance genes (tet(B), tet(L), tet(M), tet(O), tet(Q), tet(W), erm(B), erm(F) and sul2) were investigated at harvest in the leek and soil samples. In the leek samples, none of the antibiotic resistance genes were detected. In the soil samples fertilized with pig slurry, the genes erm(B), erm(F), tet(M), sul2, tet(W) and tet(O) were detected in significantly higher copy numbers in the lincomycin treatment as compared to the other antibiotic treatments. This could be due to a shift in soil microbiota induced by the addition of lincomycin. The results of this study indicate that consumption of leek carries a low risk of exposure to antibiotic residues or antibiotic resistance to doxycycline, sulfadiazine or lincomycin.
The vast majority of medicines in pig rearing are administered via oral group medication through medicated feed and drinking water. However, relevant on-farm factors affecting the concentration of ...these drugs in feed and drinking water, such as the homogeneity, stability, and cross-contamination, are largely unknown. To characterize these factors, samples of medicated feed and drinking water were taken on 24 Belgian pig farms during treatment and 2 days thereafter, as well as at different on-farm sampling sites from production to feeding troughs or drinking nipples. The samples contained amoxicillin, doxycycline, florfenicol, or flubendazole. Additionally, a questionnaire was completed. In contrast to the results of medicated feed, results of medicated water showed a large between-farm variation in antimicrobial drug concentration. The therapeutic concentration range was only met in 2 out of 11 farms using medicated feed, and in 3 out of 13 farms using medicated water. Medicated feed concentrations were often below the therapeutic concentration range mentioned in the Summary of Product Characteristics, while drinking water concentrations were just as often above as they were below the advised target concentration range. Drug residues measured 2 days after the end of therapy with both feed and water medication rarely exceeded 1% of the lowest therapeutic concentration. This study demonstrates that recommendations on good clinical practices for oral group medication in the pig industry are highly needed.
A method is described which permits the quantitative detection of the chemical coccidiostats halofuginone, robenidine, diclazuril, nicarbazin and dimetridazole and its main metabolite ...2-hydroxydimetridazole in poultry eggs and feed. Sample preparations were kept very simple and are based upon extraction with an organic solvent. Sample extracts were injected into the liquid chromatography tandem mass spectrometry (LC-MS/MS) system on a C18 column and a gradient elution was performed. Dimetridazole-D3 and diclazuril-bis, a structural analogue of diclazuril, were used as internal standards. Detection was performed on a triple quadrupole mass spectrometer in the selected reaction monitoring mode after ionisation in the positive or negative electrospray ionisation mode. Argon was applied as collision gas for collision induced dissociation. Validation of the methods was performed based on Commission Decision 2002/657/EC Official Journal of the European Communities L221 (2002) 8.
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