Short communication: Drug residues in goat milk after prophylactic use of antibiotics in intravaginal sponges for estrus synchronization.

The aim of this study was to determine whether the prophylactic use of antibiotics in intravaginal sponges used for estrus synchronization in goats may result in the presence of inhibitors in milk and, therefore, of positive results by microbial screening tests. Ninety-eight Murciano-Granadina goats were used, divided into 7 groups of 14 animals. Intravaginal sponges were placed in 6 groups using 2 concentrations of 3 different antibiotics: doxycycline, oxytetracycline, and sulfathiazole-framycetin. The sponges of the control group were placed without antibiotics. Milk samples were collected daily until 7 d posttreatment and analyzed using 3 microbial tests. Positive samples were retested by specific receptor-binding assays to confirm the positive results. Vaginal status was evaluated by visual assessment of the external aspect of the sponges after removal. The microbial test response was not affected by either day posttreatment or dose of antibiotic used, except for oxytetracycline at the higher concentration. Moreover, no positive results were obtained using receptor-binding assays, suggesting that residues, if present in milk, did not exceed the regulatory (safety) levels established for these drugs. The occurrence of soiled sponges was higher in the control group. With respect to the dose of antibiotics used, no significant differences were found for the lower dose administered. However, a significant increase in the percentage of clean sponges was observed for the higher dose of doxycycline. We conclude that the prophylactic use of low doses of doxycycline, oxytetracycline, or sulfathiazole in intravaginal sponges used for synchronization of estrus helps to reduce clinical vaginitis in dairy goats and does not seem to be the cause of positive results in microbial inhibitor tests used to detect antibiotics in goat milk.

Validation of receptor-binding assays to detect antibiotics in goat's milk.

The suitability of different receptor-binding assays to detect antibiotics in raw goat's milk was investigated. Detection capability of most ß-lactams and tetracyclines assessed applying the Betastar Combo, the SNAP Betalactam, the SNAP Tetracycline, and the Twinsensor tests was at or below maximum residue limits established by European legislation. Regarding test specificity, cross-reactions with antibiotics other than ß-lactams and tetracyclines were not found, and no false-positive results were obtained for the Betastar Combo and the SNAP tests when bulk samples of goat's milk were analyzed. For the Twinsensor test, the false-positive rate was 1%. The performance of the Betastar Combo and the SNAP tests was practically unaffected by the milk quality parameters using individual samples of goat's milk collected at points throughout the entire lactation period (false-positive rate, ≤5%). However, a larger number of positive results were obtained by the Twinsensor test in this type of milk sample (>10%), especially in the last weeks of lactation. Interferences related to the use of the preservative azidiol were not observed in any case. Neither were any significant differences found in relation to the interpretation method (visual versus instrumental) applied. In general, the response of the Betastar Combo, SNAP, and Twinsensor tests was optimal for the analysis of bulk caprine milk; thus, they may be used to monitor milk for the presence of ß-lactam and tetracycline residues in quality control programs.

[Calculation of therapeutic intensity for piglets and fattening pigs during the use of antibiotics in feed]. / Berechnung der Therapieintensität bei Ferkeln und Mastschweinen beim Einsatz von Antibiotika in Fütterungsarzneimitteln.

Oral treatments for groups of pigs via medicated feed must be prescribed on specific forms submitted to veterinary authorities. We analyzed 869 such prescription forms for the year 2009 representing the treatment of 69'863 piglets and 31'506 fattening pigs. Parameters under investigation were indication and quantity of antimicrobials prescribed. Most of the treatments took place at weaning with 10 - 14 kg and again at the beginning of the fattening period with 20 - 29 kg body weight. The average body weights at treatment were 16 kg (piglets) and 29 kg (fatteners). In analogy with the indicator DID of human medicine, we developed PIDvet describing the prescribed dose per 1000 individuals on given day in veterinary medicine. Calculated PIDvet on a given day was 180.9 for the whole population, 297.6 for piglets and 83.2 for fatteners. This shows PIDvet to be useful to stratify therapeutic intensity in different age classes and could represent a new tool to monitor the use of antibiotics.

Les aliments médicamenteux pour traiter les porcs nécessitent une prescription vétérinaire du médicament, au moyen d'un formulaire d'ordonnance officiel. Un échantillon de 869 ordonnances pour le traitement de 69'863 porcelets et de 31'506 porcs d'engraissement en 2009 a été analysé quant à l'indication et à la quantité de l'antibiotique utilisé. C'est le plus souvent lors du sevrage, avec un poids de 10 ­ 14 kg (porcelets), et lors du changement de porcherie, avec un poids de 20 ­ 29 kg (porcs d'engraissement) que les animaux sont traités. Les poids moyens dans les deux sous-populations étaient de 16 kg (porcelets) et 29 kg (porcs d'engraissement). Pour la première fois en Suisse, on a développé une mesure spécifique pour le calcul de l'intensité thérapeutique dans des groupes d'animaux, analogue à la valeur DID de la médecine humaine. Ce PIDvet décrit la somme des doses journalières pour 1000 individus par jour (prescribed daily doses per 1000 Individuals on a given day in veterinary medicine). Pour une valeur de PIDvet de 180.9 pour l'ensemble de la population à un jour donné, les valeurs individuelles pour la population de porcelets étaient de 297.6 et de 83.2 pour la population de porcs d'engraissement. Ceci montre que l'intensité thérapeutique doit être analysée de façon ciblée et par couche selon le poids et ou l'âge des animaux.

Evaluation of the Charm maximum residue limit ß-lactam and tetracycline test for the detection of antibiotics in ewe and goat milk.

The Charm maximum residue limit ß-lactam and tetracycline test (Charm MRL BLTET; Charm Sciences Inc., Lawrence, MA) is an immunoreceptor assay utilizing Rapid One-Step Assay lateral flow technology that detects ß-lactam or tetracycline drugs in raw commingled cow milk at or below European Union maximum residue levels (EU-MRL). The Charm MRL BLTET test procedure was recently modified (dilution in buffer and longer incubation) by the manufacturers to be used with raw ewe and goat milk. To assess the Charm MRL BLTET test for the detection of ß-lactams and tetracyclines in milk of small ruminants, an evaluation study was performed at Instituto de Ciencia y Tecnologia Animal of Universitat Politècnica de València (Spain). The test specificity and detection capability (CCß) were studied following Commission Decision 2002/657/EC. Specificity results obtained in this study were optimal for individual milk free of antimicrobials from ewes (99.2% for ß-lactams and 100% for tetracyclines) and goats (97.9% for ß-lactams and 100% for tetracyclines) along the entire lactation period regardless of whether the results were visually or instrumentally interpreted. Moreover, no positive results were obtained when a relatively high concentration of different substances belonging to antimicrobial families other than ß-lactams and tetracyclines were present in ewe and goat milk. For both types of milk, the CCß calculated was lower or equal to EU-MRL for amoxicillin (4 µg/kg), ampicillin (4 µg/kg), benzylpenicillin (≤ 2 µg/kg), dicloxacillin (30 µg/kg), oxacillin (30 µg/kg), cefacetrile (≤ 63 µg/kg), cefalonium (≤ 10 µg/kg), cefapirin (≤ 30 µg/kg), desacetylcefapirin (≤ 30 µg/kg), cefazolin (≤ 25 µg/kg), cefoperazone (≤ 25 µg/kg), cefquinome (20 µg/kg), ceftiofur (≤ 50 µg/kg), desfuroylceftiofur (≤ 50µg/kg), and cephalexin (≤ 50 µg/kg). However, this test could neither detect cloxacillin nor nafcillin at or below EU-MRL (CCß >30 µg/kg). The CCß for tetracyclines was also lower than EU-MRL for chlortetracycline (ewe milk: ≤ 50 µg/kg; goat milk: 75 µg/kg), oxytetracycline (≤ 50 µg/kg), and tetracycline (≤ 50 µg/kg). Regarding the 4-epimers of these tetracyclines only 4-epioxytetracycline was detected by the Charm MRL BLTET test below EU-MRL (ewe milk: 75 µg/kg; goat milk: ≤ 50 µg/kg). Acidiol had no effect on the performance of the test. The Charm MRL BLTET test could be used routinely with adapted test procedure for the fast screening of ewe and goat milk.

Thermodynamic analysis of the thermal stability of sulphonamides in milk using liquid chromatography tandem mass spectrometry detection.

The present study investigates the kinetics of the degradation of eight sulphonamides in skimmed milk when heated at 60, 70, 80, 90 and 100°C using an LC-MS/MS methodology. To determine the thermal stability of these compounds, the first-order kinetic model was applied and the activation energies, half-lives and degradation percentages were calculated. Application of kinetic equations to the different heat treatments used in dairy processing indicates that sulphonamides are very stable during pasteurisation (63°C; 30 min and 72°C; 15s) as well as UHT sterilisation (140°C; 4s). In contrast, the calculations performed with the kinetic model estimated losses in concentrations between 6.5% (sulfadimethoxine) and 85.1% (sulfamethazine) for the sterilisation at 120°C for 20 min. The existence of thermodynamic compensation was also tested for sulphonamide degradation. Results show that enthalpy and entropy values displayed a good linear relationship, and thermodynamically we can establish that the thermal degradation of sulphonamides in skimmed milk exhibits enthalpy-entropy compensation.

Use chemometric techniques in the optimization of a specific bioassay for betalactams in milk.

AIMS: A microbiological bioassay using Geoacillus stearothermophilus was optimized to detect betalactams at concentrations near to the Maximum Residue Limits (MRLs), with low cross-specificity for tetracycline. METHODS AND RESULTS: A factorial design (3 × 4) was used to evaluate the effects of concentration of spores (2·0 × 10(6), 4·0 × 10(6) and 8·0 × 10(6) spores ml(-1) ) and incubation time (3·0, 3·5, 4·0 and 4·5 h) on the response of the bioassay. Then, desirability function to raise the detection capabilities (CC(ß) ) of tetracyclines and increase sensitivity to betalactams was implemented. Significant effects of Log[S] and incubation time [It] on the CC(ß) of betalactams and tetracyclines were observed. Finally, high value of global desirability (D = 0·853), adequate betalactams CC(ß) (3·8 µg l(-1) of penicillin 'G', 27 µg l(-1) of oxacillin, 8·1 µg l(-1) of ampicillin, 48 µg l(-1) of cloxacillin) and high tetracyclines CC(ß) (5260 µg l(-1) chlortetracycline, 1550 µg l(-1) of oxytetracycline, 1070 µg l(-1) of tetracycline) were calculated. CONCLUSIONS: The application of chemometric tools allows the optimization of a bioassay that detects betalactam residues in milk. The more robust conditions have been achieved in Log[S] = 6·30 and [It] = 4·20 h. SIGNIFICANCE AND IMPACT OF THE STUDY: The logistic regression model and the desirability function are adequate chemometric techniques to improve the properties of the methods, because it is possible to increase sensitivity and decrease cross-specificity simultaneously.

Effect of heat treatments on stability of ß-lactams in milk.

The presence of residues of antimicrobial substances in milk may have serious toxicological and technical consequences. To date, few studies have been done to evaluate the effect of heat treatments on ß-lactam residues in milk. However, the few studies that have been conducted estimate losses of antimicrobial activity under different combinations of temperature and time using microbiological methods. The aims of this study were to calculate the kinetic parameters for the degradation of ß-lactam antibiotics in milk and to develop prediction models to estimate the concentration losses of these compounds in conventional dairy heat treatments. To do so, we employed a quantitative HPLC method to calculate losses in concentrations of 10 ß-lactam antibiotics in milk with different combinations of temperature and time. Increasing the temperature from 60°C to 100°C decreased the half-life of amoxicillin (372 to 50 min), ampicillin (741 to 26 min), cloxacillin (367 to 46 min), and penicillin G (382 to 43 min). These increases in temperature caused further degradation in cephalosporins, which was accompanied by a decrease in half-life times to reach very low values; for instance, 4, 5, and 6 min for cefoperazone, cephurexime, and cephapirin, respectively. Kinetic equations were applied to different heat treatments used in dairy processing. Heat treatments at high temperatures and long times (e.g., 120°C for 20 min) led to a further degradation of ß-lactam antibiotics with percentages close to 100% for cefoperazone and cefuroxime. In contrast, when milk was subjected to heat treatments at lower temperatures and times (e.g., 72°C for 15s), the degradation of ß-lactam in milk did not exceed 1% for the 10 antibiotics tested.

Heat treatment effects on the antimicrobial activity of macrolide and lincosamide antibiotics in milk.

Antibiotic residues in milk can cause serious problems for consumers and the dairy industry. Heat treatment of milk may diminish the antimicrobial activity of these antibiotic residues. This study analyzed the effect of milk processing (60 °C for 30 min, 120 °C for 20 min, and 140 °C for 10 s) on the antimicrobial activity of milk samples fortified with three concentrations of three macrolides (erythromycin: 20, 40 and 80 µg/liter; spiramycin: 100, 200, and 400 µg/liter; and tylosin: 500, 1,000, and 2,000 µg/liter) and one lincosamide (lincomycin: 1,000, 2,000, and 4,000 µg/liter). To measure the loss of antimicrobial activity, a bioassay based on the growth inhibition of Micrococcus luteus was done. The data were analyzed using a multiple linear regression model. The results indicate that treatment at 120 °C for 20 min produces inactivation percentages of 93% (erythromycin), 64% (spiramycin), 51% (tylosin), and 5% (lincomycin), while treatment at 140 °C for 10 s results in generally lower percentages (30% erythromycin, 35% spiramycin, 12% tylosin, and 5% lincomycin). The lowest loss or lowest reduction of antimicrobial activity (21% erythromycin and 13% spiramycin) was obtained by treatment at 60 °C for 30 min.

Optimization of bioassay for tetracycline detection in milk by means of chemometric techniques.

AIMS: In this study, a microbiological method of dichotomous response using Bacillus cereus was designed and optimized to detect tetracyclines (TCs) at concentrations near to the maximum residue limits (MRLs). METHODS AND RESULTS: In a first stage, the response time of bioassay was reduced to 5 h when the logarithm of spore concentration (log S) was increased. Later, a Plackett Burman design (2(6-3)) was analysed using logistic regression model. This design indicates significant effects of log S and chloramphenicol (CAP) on the detection limit (DL) of TC. Then, the response surfaces (RS) of the TCs DTs as a function of log S and CAP were plotted using a Dohlert design and the logistic regression model. These RS show a linear decrease with the raise of CAP and a quadratic effect of log S. Finally, the DTs of TC (109 µg l(-1)) and oxytetracycline (100 µg l(-1)) were adjusted to their MRLs through the desirability function. CONCLUSIONS: By successive application of experimental design techniques could be optimized a bioassay for the detection of TC residues in milk. The best conditions have been achieved when the assay was made with log S = 5·12 and CAP = 470 µg l(-1). SIGNIFICANCE AND IMPACT OF THE STUDY: Experimental design techniques together with the logistic regression model and the desirability function represent an adequate tool for the optimization of a bioassay with binary response.

Effect of heating on the stability of quinolones in milk.

Nowadays, the possible public health risk associated with the presence of quinolone residues and other antibiotics in milk is well-known, but there is a lack of information about the effect milk processing temperatures have on the presence of antimicrobial residues. The aim of this work was to determine the effect of different temperatures and heating times on the concentration of quinolones in milk by employing liquid chromatographic equipment analysis with fluorescence detection. In order to determine the thermo-stability of these compounds, the first-order kinetic model was applied, and the activation energies, half-lives, and percentages of degradation of each compound were calculated. Results showed that quinolones are very resistant to different heat treatments with maximum losses of concentration of 12.71% for ciprofloxacin and 12.01% for norfloxacin at 120 degrees C and 20 min. The high stability of quinolones represents a significant risk to human health because the residues of these antibiotics can remain in milk after heat treatment and, therefore, can reach the dairy industry and consumers.

Effect of heat treatments on aminoglycosides in milk.

The presence of antibiotic residues in milk not only is a potential consumer risk but also may cause serious problems in the fermentation processes used in the dairy industry. There is very limited information available on the effect of heat treatments on aminoglycoside activity in milk. For this reason, the objective of this study was to analyze the effect of different heat treatments (60 degrees C for 30 min, 120 degrees C for 20 min, and 140 degrees C for 10 s) on milk samples spiked with four aminoglycosides (gentamicin, 50, 100, and 200 microg/liter; kanamycin, 300, 600, and 1200 microg/liter, neomycin, 200, 400, and 800 microg/liter; and streptomycin, 200, 400, and 800 microg/liter). The method used was a bioassay based on the inhibition of Bacillus subtilis BGA. Statistical analysis of the three heat treatments studied showed that the one at 60 degrees C for 30 min did not inactivate the aminoglycosides, the treatment at 140 degrees C for 10 s produced inactivation levels of between 17% for kanamycin and 40% for neomycin, and the classic sterilization (120 degrees C for 20 min) showed a high heat inactivation (>95%) for all the concentrations of aminoglycosides tested with respect to the samples without treatment (control group).

Robust experimental design for optimizing the microbial inhibitor test for penicillin detection in milk.

AIMS: To use experimental design techniques and a multiple logistic regression model to optimize a microbiological inhibition test with dichotomous response for the detection of Penicillin G in milk. METHODS AND RESULTS: A 2(3) x 2(2) robust experimental design with two replications was used. The effects of three control factors (V: culture medium volume, S: spore concentration of Geobacillus stearothermophilus, I: indicator concentration), two noise factors (Dt: diffusion time, Ip: incubation period) and their interactions were studied. The V, S, Dt, Ip factors and V x S, V x Ip, S x Ip interactions showed significant effects. CONCLUSIONS: The use of 100 microl culture medium volume, 2 x 10(5) spores ml(-1), 60 min diffusion time and 3 h incubation period is recommended. In these elaboration conditions, the penicillin detection limit was of 3.9 microg l(-1), similar to the maximum residue limit (MRL). Of the two noise factors studied, the incubation period can be controlled by means of the culture medium volume and spore concentration. SIGNIFICANCE AND IMPACT OF THE STUDY: We were able to optimize bioassays of dichotomous response using an experimental design and logistic regression model for the detection of residues at the level of MRL, aiding in the avoidance of health problems in the consumer.

Heat inactivation of beta-lactam antibiotics in milk.

The presence of residues of antimicrobial substances in milk is one of the main concerns of the milk industry, as it poses a risk of toxicity to public health, and can seriously influence the technological properties of milk and dairy products. Moreover, the information available on the thermostability characteristics of these residues, particularly regarding the heat treatments used in control laboratories and the dairy industry, is very scarce. The aim of the study was, therefore, to analyze the effect of different heat treatments (40 degrees C for 10 min, 60 degrees C for 30 min, 83 degrees C for 10 min, 120 degrees C for 20 min, and 140 degrees C for 10 s) on milk samples fortified with three concentrations of nine beta-lactam antibiotics (penicillin G: 3, 6, and 12 microg/liter; ampicillin: 4, 8, and 16 microg/liter; amoxicillin: 4, 8, and 16 microg/liter; cloxacillin: 60, 120, and 240 microg/liter; cefoperazone: 55, 110, and 220 microg/liter; cefquinome: 100, 200, and 400 microg/liter; cefuroxime: 65, 130, and 260 microg/liter; cephalexin: 80, 160, and 220 microg/ liter; and cephalonium: 15, 30, and 60 microg/liter). The method used was a bioassay based on the inhibition of Geobacillus stearothermophilus var. calidolactis. The results showed that heating milk samples at 40 degrees C for 10 min hardly produced any heat inactivation at all, while the treatment at 83 degrees C for 10 min caused a 20% loss in penicillin G, 27% in cephalexin, and 35% in cefuroxime. Of the three dairy industry heat treatments studied in this work, low pasteurization (60 degrees C for 30 min) and treatment at 140 degrees C for 10 s only caused a small loss of antimicrobial activity, whereas classic sterilization (120 degrees C for 20 min) showed a high level of heat inactivation of over 65% for penicillins and 90% for cephalosporins.

Screening of antibiotic residues in ewes' milk destined to cheese by a commercial microbiological inhibition assay.

Bulk ewes' milk from Spanish dairy farms in the Castilla-La Mancha region and destined for production of protected denomination of origin (PDO) Manchego cheese were analysed each month for antimicrobial residues during the course of 1 year. A microbiological assay specific for ewes' milk (Eclipse '100ov') was used. The number of positive samples by the Eclipse '100ov' test was 2.6%. A second examination of positives following heat treatment at 82 degrees C for 10 min showed a reduction of up to 0.9% (i.e. 63% of the samples detected corresponded to 'false-positives'). Of the confirmed positives, 25% were identified as beta-lactams by a penicillinase solution; the remainder corresponded to antimicrobial compounds that could not be identified. The month of collection was related to the probability of positive results, but to no other factors such as somatic cell counts, bacteriology or composition (fat and protein). The highest rates of positives were observed in September and October. Finally, the results were compared with those obtained by the Delvotest SP method, which showed the detection of a higher number of positive samples compared with the Eclipse '100ov' method.

[CliniTox: the computer-based information system for poisoning in farm animals]. / CliniTox: Das computergestützte Informationssystem für Vergiftungen beim Nutztier.

Poisonings can cause reduced performance and severe economic loss in farm animals. A swift and targeted action is required from veterinarians. We have established a computer-based decision support system for poisonings in ruminants and pigs. The system offers access to the most recent information available in clinical toxicology of farm animals. Towards this goal, relevant reports from the peer-reviewed literature were evaluated and organised according to the requirements of a structured database. The information provided for each toxic substance includes a summary of its chemical and physical properties, sources, commercial uses or natural occurrences, toxicokinetic data, mechanisms of action, threshold doses, clinical symptoms with brief case reports, sampling and analytical results, post-mortem abnormalities, differential diagnoses, therapeutic guidelines and references to the literature. This decision support system has been programmed with two user-friendly search functions: a search tool that allows the choice of clinical and pathological findings, and another function that serves to find a substance using its chemical name, the class of compounds to wich it belongs, a possible source or one of its main applications. CliniTox can be accessed directly via our webserver (http://www.clinitox.ch).

Occurrence of antibiotic residues in milk from Manchega ewe dairy farms.

Ewe milk samples from different ovine dairy farms from the Castilla-La Mancha region of Spain were collected from bulk tanks to estimate the occurrence of antibiotic residues in raw and heated (82 degrees C, 10 min) milk by the Delvotest SP test. The month of collection, somatic cell counts, and bacteriology were analyzed and examined by means of a logistic regression model. The screening of a total of 2686 raw milk samples showed 1.7% "positive" and 2.1% "doubtful" results, which decreased after heating treatment to 1.3% and 0.4%, respectively. "Positive" and "doubtful" samples were identified by penicillinase and p-aminobenzoic acid solutions, and the majority of them corresponded to antimicrobials different than beta-lactams or sulfonamides. By applying a logistic regression model, a significant effect of month of collection and bacteriology was observed in the initial screening and after the heat treatment. The highest percentages of "positive plus doubtful" results were observed in late summer-early autumn. A slight peak was also observed in spring in raw milk samples. Bacteriology was positively correlated with "positive plus doubtful" results.

Performances of antibiotic screening tests in determining the persistence of penicillin residues in ewe's milk.

Milk collected at 12-h intervals throughout 6 days from three groups of Manchega ewes (n = 12 per group), treated intramuscularly with beta-lactams (benzyl penicillin procaine, ampicillin, and amoxicillin, respectively), was evaluated for antibiotic residue persistence with four microbial inhibitor tests (BRT MRL, CH ATK P&S, Delvotest SP, and Eclipse 100ov) and one enzymatic test (Penzym 100). Antibiotic depletion time was established using a logistic regression model. A clear effect (P < 0.0001) of milking order on the response of all tests was observed with the three antibiotics, but no significant effects were found for milk yield. Except with Eclipse 100ov, positive tests were observed after the recommended withdrawal period of benzyl penicillin procaine (five milkings) from 2% (Delvotest SP) to 11% (CH ATK P&S). There were almost no positive responses beyond the withdrawal period (six milkings) of ampicillin, except for the Penzym 100 test (7%). Residues of amoxicillin were found to persist beyond the six milkings established as the withdrawal period, from 2.8% (Eclipse 100ov) to 72.4% (CH ATK P&S) of positive cases. Higher frequencies of doubtful cases were found with BRT MRL and Delvotest SP assays with the three beta-lactams. Positive and doubtful results could be obtained when milk samples from individual ewes were analyzed using BRT MRL, CH ATK P&S, Delvotest SP, and Penzym 100 tests, even if farmers follow the antibiotic withdrawal periods.

Evaluation of screening test for detection of antimicrobial residues in ewe milk.

The effects of preservatives (potassium dichromate and sodium azide), heat treatment (untreated and 82 degrees C/10 min), and lactation stage upon the response of the microbial tests (BRT AiM and Delvotest) utilized for the detection of residues of antimicrobial substances in ewe milk were examined. Milk samples were collected from the morning milking of 50 Manchega ewes every 2 wk, from 15 d postpartum until the end of lactation. A total of 2322 samples were analyzed by BRT AiM with prediffusion and Delvotest microbial tests. The specificity of preservative-free milk samples without heat treatment was high (96.3% for BRT and 97.7% for Delvotest), with results improving for those samples thermally treated at 82 degrees C/10 min (99.0% for BRT and 98.7% for Delvotest). Potassium dichromate produced a total inhibition of growth of Bacillus stearothermophilus with both methods. When acidiol was utilized, the specificity of the samples not treated thermally was lower compared with preservative-free milk samples for the BRT AiM (90.2%) and Delvotest (91.0%) methods, improving when the samples were thermally treated, both for BRT AiM (94.8%) and Delvotest (95.3%), given that the presence of the preservative increased the frequency of doubtful results. The lactation stage significantly affected the results of the methods, with a greater frequency of false-positive and doubtful cases toward the end of the cycle, especially in those samples preserved with acidiol. The greater selectivity in both methods was therefore obtained for preservative-free ewe milk samples with prior heat treatment taken at the beginning or in midlactation period.

Detection limits of antimicrobials in ewe milk by delvotest photometric measurements.

The Delvotest method detection limits per manufacturer's instructions at a fixed reading time of 3 h for 24 antimicrobial agents were determined in ewe milk by photometric measurement. For each drug, eight concentrations were tested on 20 ewe milk samples from individual ewes. Detection limits, determined by means of logistic regression models, were (microg/kg): 3, amoxycillin; 2, ampicillin; 18, cloxacillin; 1, penicillin "G"; 34, cefadroxil; 430, cephalosporin "C"; 40, cephalexin; 20, cefoperazone; 33, Ceftiofur; 18, cefuroxime; 6100, streptomycin; 1200, gentamycin; 2600, neomycin; 830, erythromycin; 100, tylosin; 180, doxycycline; 320, oxytetracycline; 590, tetracycline; 88, sulfadiazine; 44, sulfamethoxazole; 140, sulfametoxypyridazine; 48, sulfaquinoxaline; 12,000, chloramphenicol; and 290, trimethoprim. Whereas the beta-lactam antibiotics, sulphonamides, and tylosin were detected by Delvotest method at levels equal to those of maximum residue limits, its sensitivity needs to be enhanced to detect aminoglycosides, tetracyclines, streptomycin, chloramphenicol, and trimethoprim residues in ewe milk or to develop an integrated residue detection system for ewe milk with different sensitive microorganisms for each group of antiinfectious agents.

Residue persistence in sheep milk following antibiotic therapy.

Drug residues in milk supplies may have public health implications and can interfere in the manufacture of dairy products, such as cheese. In Spain, most ewe milk production is destined for cheese making, often using raw milk. This study analyses the main factors influencing antibiotic depletion time in lactating dairy sheep. 42, Manchega ewes were distributed into three groups, each receiving a different treatment (cephalexin intramammary infusion, penicillin G intramuscular, and oxytetracycline intravenous injections). During and after the recommended withdrawal period, milk samples were taken at each milking. A microbiological inhibition test (Brilliant Black Reduction, BRT) was used to screen all samples and antibiotic withholding times were established using a logistic regression model. The response to the BRT method in milk from individual ewes treated showed that the effect of the milking order was significant (P<0.001) with the three antibiotics. However the only influence on milk yield was with the intramammary treatment (P<0.005). The BRT method was found to be very sensitive, particularly to the two beta-lactamic antibiotics.