Modified microbiological method for the screening of antibiotics in milk.

The Bacillus stearothermophilus disc assay is routinely used by the dairy industry to screen milk for antibiotic residues. Although the assay detects the presence of beta-lactam antibiotics, it does not distinguish cephalosporins from other beta-lactam antibiotics. In this study, the B. stearothermophilus disc assay was modified to allow it to distinguish parent ceftiofur from other antibiotics by incorporation of the enzymes penicillinase and cephalosporinase into the assay. The modified B. stearothermophilus disc assay involves determining the zone of inhibition of a sample on an agar plate after the plate was incubated at 65 degrees C for 2.5 to 3 h as well as determining the zone of inhibition after the sample was treated with penicillinase or cephalosporinase. Samples in which this zone diameter was > 19 mm and < or = 25 mm were interpreted using the data from the primary assay. Samples with zone diameters > 25 mm must be diluted 2- to 10-fold and reassayed to obtain a zone diameter > 19 and < or = 25 mm, for proper interpretation. Samples with zone diameters > or = 16 mm and < or = 19 mm must also be reassayed using dilute enzyme solutions for proper interpretation. When these modifications of the B. stearothermophilus disc assay are used, ceftiofur can be distinguished from ampicillin, amoxicillin, penicillin, cephapirin, cloxacillin, novobiocin, and pirlimycin for samples with zone diameters > or = 16 mm. This assay cannot, however, separate ceftiofur from cefazolin.(ABSTRACT TRUNCATED AT 250 WORDS)

Concentration of ceftiofur metabolites in the plasma and lungs of horses following intramuscular treatment.

Ceftiofur sodium, a broad spectrum cephalosporin antibiotic approved for veterinary use, is metabolized to desfuroylceftiofur which is conjugated to micro as well as macromolecules. Twelve horses, weighting 442-618 kg, were injected intramuscularly with a single dose of 2.2 mg ceftiofur/kg (1.0 mg/lb) body weight. Blood was collected at various intervals over 24 h after treatment. Three groups of four horses each were euthanized and lungs were collected at 1, 12, and 24 h after treatment. The concentration of desfuroylceftiofur and desfuroylceftiofur conjugates in the plasma and lungs was determined by converting them to desfuroylceftiofur acetamide (DCA) and measured DCA by high performance liquid chromatography with UV detection. The average maximum concentration (Cmax) of desfuroylceftiofur and related metabolites in plasma expressed as ceftiofur equivalents was 4.46 +/- 0.93 micrograms/ml occurred at 1.25 +/- 0.46 h after treatment. These concentrations declined to 0.99 +/- 0.16, 0.47 +/- 0.15 and 0.17 +/- 0.02 microgram/ml at 8, 12, and 24 h, respectively. The mean residence time of ceftiofur metabolites was 6.10 +/- 1.27 h. Concentrations of desfuroylceftiofur and desfuroylceftiofur conjugates in the lungs of horses expressed as ceftiofur equivalents were 1.40 +/- 0.36, 0.27 +/- 0.07, and 0.15 +/- 0.08 micrograms/ml at 1, 12, and 24 h, respectively. These concentrations of the drug at 12 and 24 h in lung homogenate were similar but slightly lower than plasma concentrations in the same horses, and the plasma pharmacokinetic values including half-life were similar to those observed at the approved dose of 1.1-2.2 mg ceftiofur/kg body weight administered intramuscularly once daily for 3-5 days in cattle.

Depletion of intramuscularly injected ceftiofur from the milk of dairy cattle.

Ceftiofur sodium, a new broad-spectrum cephalosporin, has been approved in the US, Canada, and several other countries throughout the world to treat bovine respiratory disease in cattle and dairy cows. In Experiment 1, 6 lactating cows were intramuscularly treated with 2.29 mg of [14C]ceftiofur/kg of BW daily for 5 d. In Experiment 2, 30 additional cows at three locations were similarly treated with 2.2 mg of ceftiofur (unlabeled)/kg of BW. Milk was collected every 12 and 24 h after each dose and every 12 h up to 5 d after the last dose. The majority of milk samples, both during treatment (12 and 24 h after each dose) and after the last dose (up to 5 d following ceftiofur treatment), were negative by screening procedures based on microbial inhibition (Delvotest-P, Bacillus stearothermophilus disk assay, and cylinder plate assays). The receptor-binding Charm Test II assay, which has a limit of detection of .005 ppm of ceftiofur, gave positive tests for milk samples up to 48 h following treatment. When the Charm Test II assay is used with .008 IU/ml of penicillin as a positive control, 44% of the samples from individual cows were negative at 12 h posttreatment. Ninety percent of the samples from individual cows were negative at 24 h after the last treatment. The use of ceftiofur in dairy cattle in accordance with the label directions does not result in total residues in milk higher than the FDA-calculated safe concentration of 1-ppm ceftiofur equivalents. The milk from individual cows did not test positive by the commercial screening assays examined in this study, except for the Charm Test II. The Charm Test II was 90% negative using the Charm Sciences criteria at 24 h after the last treatment.

Tissue concentrations of clindamycin after multiple oral doses in normal cats.

Eighteen normal cats were randomly allocated into two blocks with three treatment groups and dosed orally with clindamycin aqueous solution for 10 days at a dosage rate of 5.5 mg/kg twice daily (Group 1), 11 mg/kg twice daily (Group 2), or 22 mg/kg once daily (Group 3). At the end of dosing, all cats were killed and tissues were taken for clindamycin concentration analysis. Clindamycin was extracted from tissues using solid-phase extraction columns followed by microbiological assay of clindamycin using a cylinder plate assay using M. luteus. Recovery from each tissue was determined by inoculating known concentrations of clindamycin into drug-naive tissues and comparing the observed concentration from the expected concentration. Confirmation that the bioassay detected clindamycin and not N-desmethylclindamycin, its active metabolite, was done using gas-chromatography-mass-spectrometry. Concentrations were highest in the lung, with tissue:serum ratios greater than 3 in all groups. Concentrations were higher in Group 3 than Group 1 (P less than 0.05). Only liver concentrations in Group 3 were statistically higher than in Group 2, although all tissues except bone marrow and CSF had numerically higher concentrations in Group 3 than Group 2. The tissue:serum ratio was greater than 1 in all tissues studied except bone, cerebrospinal fluid, brain, and skeletal muscle.

Human neutrophil activation with interleukin-1. A role for intracellular calcium and arachidonic acid lipoxygenation.

Human monocyte-derived interleukin-1 (IL-1) stimulated the selective extracellular release of cytoplasmic granule-associated elastase from human neutrophils. Although extracellular calcium (Ca2+) enhances but is not required for the expression of granule exocytosis, IL-1 did induce the mobilization of previously sequestered intracellular Ca2+ as measured with the highly selective fluorescent Ca2+ indicator, Quin 2. Further, IL-1 stimulated the mobilization of cell membrane-associated Ca2+ as monitored by a decrease in fluorescence of chlorotetracycline (CTC)-loaded neutrophils. W-7, a calmodulin antagonist, and TMB-8[8(N,N-diethylamino)-octyl-(3,4,5-trimethoxy)benzoate hydrochloride], an intracellular Ca2+ antagonist, inhibited the Quin 2 fluorescent response by neutrophils to IL-1. TPCK (N-alpha-p-tosyl-L-lysine chloromethylketone), a serine protease inhibitor, suppressed IL-1-induced Quin 2 and CTC fluorescence. Exposure of neutrophils to IL-1 resulted in a concentration-dependent production of the 5-lipoxygenase product, LTB4 [5(S),12(R)-dihydroxy-6,14-cis-8,10-trans-eicosatetraenoic acid] which was enhanced in the presence of arachidonic acid (AA). LTB4 production by IL-1-activated neutrophils was suppressed by the lipoxygenase inhibitors nordihydroguaiaretic acid (NDGA) and piriprost potassium [6,9,deepoxy-6,9-(phenylimino)-delta 6,8-prostaglandin l1], and a cyclooxygenase/lipoxygenase inhibitor, 5,8,11,14-eicosatetraynoic acid (ETYA), whereas a cyclooxygenase inhibitor, flurbiprofen, was inactive. These data indicate that cytosolic free Ca2+ ([Ca2+]i) and a metabolite(s) of AA lipoxygenation mediate granule exocytosis elicited with IL-1.

A possible requirement for arachidonic acid lipoxygenation in the mechanism of phagocytic degranulation by human neutrophils stimulated with aggregated immunoglobulin G.

Aggregated immunoglobulin G (AggIgG) caused a concentration-dependent extracellular release of granule-associated lysozyme and myeloperoxidase (MPO) from human neutrophils. Generation of the 5-lipoxygenase product of arachidonic acid (AA) metabolism, 5(S),12(R)-dihydroxy-6,14-cis,8,10-trans-eicosatetraenoic acid [leukotriene B4 (LTB4)], by neutrophils is exposed to AggIgG occurred in the presence but not absence of exogenous AA. U-60,257B (piriprost potassium), an inhibitor of leukotriene synthesis, caused a dose-related suppression of LTB4 production and granule exocytosis by AggIgG-treated cells. These data suggest that a lipoxygenase product of AA metabolism may mediate AggIgG-induced phagocytic release of granule constituents from neutrophils.

Bioluminescent enzyme immunoassay for estriol. Use of reversibly inactivated bacterial luciferase as label.

A bioluminescent enzyme immunoassay using estriol labeled with reversibly inactivated bacterial luciferase is described. An estriol derivative bearing an alkylthiolsulfonate is linked to the cysteinyl thiols of luciferase by formation of mixed disulfide linkages; thus, luciferase becomes inactive. After immunoassay, the inactive luciferase of the label bound to the immunoprecipitate is reactivated by incubation with dithiothreitol and the luciferase activity then is quantitated by a 20-s reaction performed with an automated luminometer (LKB 1251). Under the defined conditions, the labels are stable for at least 14 days as tested at 4 degrees C. A standard curve with a wide linear range from 50 to 6000 pg is demonstrated. This unique technology discussed here, therefore, offers exciting possibilities as a sensitive and rapid enzyme immunoassay for estriol.