Ceftiofur Metabolites Research Articles

Excreted Antibiotics May Be Key to Emergence of Increasingly Efficient Antibiotic Resistance in Food Animal Production.

At a time when antibiotic resistance is seemingly ubiquitous worldwide, understanding the mechanisms responsible for successful emergence of new resistance genes may provide insights into the persistence and pathways of dissemination for antibiotic-resistant organisms in general. For example, Escherichia coli strains harboring a class A β-lactamase-encoding gene (blaCTX-M-15) appear to be displacing strains that harbor a class C β-lactamase gene (blaCMY-2) in Washington State dairy cattle. We cloned these genes with native promoters into low-copy-number plasmids that were then transformed into isogenic strains of E. coli, and growth curves were generated for two commonly administered antibiotics (ampicillin and ceftiofur). Both strains met the definition of resistance for ampicillin (≥32 μg/mL) and ceftiofur (≥16 μg/mL). Growth of the CMY-2-producing strain was compromised at 1,000 μg/mL ampicillin, whereas the CTX-M-15-producing strain was not inhibited in the presence of 3,000 μg/mL ampicillin or with most concentrations of ceftiofur, although there were mixed outcomes with ceftiofur metabolites. Consequently, in the absence of competing genes, E. coli harboring either gene would experience a selective advantage if exposed to these antibiotics. Successful emergence of CTX-M-15-producing strains where CMY-2-producing strains are already established, however, requires high concentrations of antibiotics that can only be found in the urine of treated animals (e.g., >2,000 μg/mL for ampicillin, based on literature). This ex vivo selection pressure may be important for the emergence of new and more efficient antibiotic resistance genes and likely for persistence of antibiotic-resistant bacteria in food animal populations. IMPORTANCE We studied the relative fitness benefits of a cephalosporin resistance enzyme (CTX-M-15) that is displacing a similar enzyme (CMY-2), which is extant in E. coli from dairy cattle in Washington State. In vitro experiments demonstrated that CTX-M-15 provides a significant fitness advantage, but only in the presence of very high concentrations of antibiotic that are only found when the antibiotic ampicillin, and to a lesser extent ceftiofur, is excreted in urine from treated animals. As such, the increasing prevalence of bacteria with blaCTX-M-15 is likely occurring ex vivo. Interventions should focus on controlling waste from treated animals and, when possible, selecting antibiotics that are less likely to impact the proximal environment of treated animals.

Faecal concentrations of ceftiofur metabolites in finisher pigs administered intramuscularly with ceftiofur.

The objective of this study was to determine the effects of dietary fibre level and source on faecal ceftiofur metabolites concentrations after intramuscular administration of therapeutic ceftiofur hydrochloride in finisher pigs. Pens of finisher pigs (n = 36), with an equal number of barrows and gilts, were randomly assigned to 1 of 3 dietary treatment groups: basal diet composed of corn grain and soy bean meal with no supplement and formulated to contain 8.7% neutral detergent fibre (NDF), supplemented with 20% distillers dried grains with solubles (a byproduct of the ethanol production from corn grain) formulated to contain 13.6% NDF, primarily insoluble fibre or supplemented with 14.5% sugar beet pulp formulated to contain 13.6% NDF. Faecal samples were collected 6–8 hr after ceftiofur injection from treated and untreated pen‐mate pigs on days 1 and 3 of the 3‐day treatment regimen. Faecal concentrations of ceftiofur metabolites, including the major metabolite, desfuroylceftiofur, were analysed by reverse‐phase high pressure liquid chromatography with ultraviolet detection. Overall, the faecal concentrations of ceftiofur metabolites did not differ significantly between the dietary treatments. The mean concentrations of metabolites tended to be lower (p = .1) on day 3 compared to day 1 of the 3‐day treatment regimen. Faecal concentrations of metabolites were not affected by the gender of the finisher pigs. The concentrations of ceftiofur metabolites in the faeces are likely reflective of the microbial activity in the hindgut. Our data suggest that the fibre level and source used in the study did not affect the faecal concentrations of ceftiofur metabolites.

BL30SEC Method for Detection of Beta-Lactams in Raw Commingled Cow Milk: AOAC Performance Tested MethodSM 061902.

Testing milk for antibiotics before acceptance into dairies is required by the U.S. Pasteurized Milk Ordinance. Technological advances in tests have reduced screening times and improved detection accuracy. This work describes the validation of the Charm Rapid One Step Assay Beta-Lactam 30 Second Test according to the U.S. Food and Drug Administration Center for Veterinary Medicine protocol for raw commingled milk. Milk is added to the lateral flow test strip in an incubator/reader to deliver a 30 second result. Independent laboratory validation followed sensitivity, interference, and incurred residue protocols. Sensitivity, in parts per billion (ppb = µg/kg), using a probit curve determined 90% percent detection with 95% confidence, which met National Conference of Interstate Milk Shipments (NCIMS) specifications. Six U.S. approved beta-lactam drugs were detected below, but within 50% of, target/tolerance levels for penicillin G 2.9 ppb, ampicillin 5.9 ppb, amoxicillin 5.8 ppb, cephapirin 13 ppb, cloxacillin 8.1 ppb, and ceftiofur metabolites 73 ppb. No interferences were observed from 33 animal drugs at 100 ppb, somatic cells at 1.2 million/mL, or bacterial levels of >300000 CFU/mL. Incurred residue detection levels were similar to levels determined with the spiked parent compound. The data support NCIMS that the BL30SEC method met U.S. criteria for testing milk for beta-lactams.

Pharmacokinetic Modeling of Ceftiofur Sodium Using Non-linear Mixed-Effects in Healthy Beagle Dogs.

Ceftiofur (CEF) sodium is a third-generation broad-spectrum cephalosporin commonly used in an extra-label manner in dogs for the treatment of respiratory and urinary system infections. To contribute to the literature supporting CEF use in companion animals, we have developed a compartmental, non-linear mixed-effects (NLME) model of CEF pharmacokinetics in dogs (PK). We then used the mathematical model to predict (via Monte Carlo simulation) the duration of time for which plasma concentrations of CEF and its pharmacologically active metabolites remained above minimum inhibitory concentrations (respiratory tract Escherichia coli spp.). Twelve healthy beagle dogs were administered either 2.2 mg/kg ceftiofur-sodium (CEF-Na) intravenously (I.V) or 2.2 mg/kg CEF-Na subcutaneously (S.C). Plasma samples were collected over a period of 72 h post-administration. To produce a measurement of total CEF, both CEF and CEF metabolites were derivatized into desfuroylceftiofur acetamide (DCA) before analysis by UPLC-MS/MS. No adverse effects were reported after I.V or S.C dosing. The NLME PK models were parameterized using the stochastic approximation expectation maximization algorithm as implemented in Monolix 2018R2. A two-compartment mamillary model with first-order elimination and first-order S.C absorption best described the available kinetic data. Final parameter estimates indicate that CEF has a low systemic clearance (0.25 L/h/kg) associated with a low global extraction ratio E = 0.02) and a moderate volume of distribution (2.97 L/kg) in dogs. The absolute bioavailability after S.C administration was high (93.7%). Gender was determined to be a significant covariate in explaining the variability of S.C absorption. Our simulations predicted that a dose of 2.2 mg/kg CEF-Na S.C would produce median plasma concentrations of CEF of at least 0.5 μg/mL (MIC50) for ~30 h.

Short communication: Use of the BetaStar Plus assay for detection of ceftiofur antimicrobial residues in milk from individual cows following intramammary treatment for mastitis

Development and use of on-farm assays to detect antimicrobial residues in milk is important to reduce the risk of violative residues in marketed milk. The objective of this study was to evaluate the effectiveness of a lateral-flow immunodiagnostic assay (BetaStar Plus, Neogen Corp., Lansing, MI) in detecting ceftiofur residues in milk from individual cows treated for mastitis. This assay is currently approved by the US Federal Drug Administration (FDA) for detecting β-lactam residues in commingled milk. Forty-five dairy cows with clinical mastitis from 4 dairy farms were enrolled and treated intramammary with 125 mg of ceftiofur hydrochloride (Spectramast LC, Zoetis, Madison, NJ) according to the manufacturer’s label recommendation. Composite milk samples were collected (A) before first intramammary antimicrobial treatment, (B) before the last intramammary antimicrobial treatment, (C) the last milking of the product-labeled milk withhold, (D) the first milking after the product-labeled milk withhold had been met, and (E) 72 h after the product-labeled milk withhold had been met. Samples were tested using the BetaStar Plus assay within 48 h of collection. Parallel samples were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and for somatic cell count and milk components. The BetaStar Plus assay identified 6.7, 60.0, 46.7, 22.2, and 6.7% positive samples at each of the respective time points. The assay had sensitivity and specificity of 100 and 84.7%, respectively, compared with liquid chromatography-tandem mass spectrometry analysis using FDA published residue tolerance levels for ceftiofur (or ceftiofur metabolites) as a threshold. The BetaStar Plus assay could be useful for detecting ceftiofur residues in milk from individual cows following intramammary treatment for mastitis before the milk is shipped for processing.

Plasma pharmacokinetics of ceftiofur metabolite desfuroylceftiofur cysteine disulfide in holstein steers: application of nonlinear mixed-effects modeling.

Eight clinically normal and drug-naïve Holstein steers were dosed with ceftiofur sodium at 2.2mg/kg body weight intramuscularly. Doses were given at 24-h intervals for 5days. Prior to the first dose and after all injections, blood samples were collected serially for determination of plasma concentrations of one of ceftiofur's main metabolites, desfuroylceftiofur cysteine disulfide (DCCD). A nonlinear mixed-effect model was used to analyze the plasma concentration data. A stochastic approximation expectation maximization (SAEM) algorithm in MONOLIX version 4.2.2 was used to approximate the likelihood of the nonlinear mixed-effect model and to estimate the population parameters. In addition, simulation studies were conducted to justify the model and demonstrate how to interpret the model parameters given different scenarios.

A metallo-β-lactamase is responsible for the degradation of ceftiofur by the bovine intestinal bacterium Bacillus cereus P41

Ceftiofur is a highly effective veterinary cephalosporin, yet it is rapidly degraded by bacteria in the gut. The goal of this work was to directly determine the mechanism of ceftiofur degradation by the bovine intestinal isolate Bacillus cereus P41. B. cereus P41 was isolated from the feces of a cow that had not been treated with cephalosporins, and was found to rapidly degrade ceftiofur in culture. Analysis of spent culture media by HPLC/UV and HPLC/MS revealed one major metabolite of ceftiofur, with a negative ion m/z of 127. Comparison of ceftiofur, ceftriaxone, and cefpodoxime degradation suggested that the major stable ceftiofur metabolite was the thiofuroic acid group eliminated from the C-3 position of the drug after hydrolysis by β-lactamase. Genomic DNA from B. cereus P41 was cloned into Escherichia coli, and the transformants were screened for growth in the presence of ceftiofur. DNA sequencing of the plasmid pHSG299-BC-3 insert revealed the presence of a gene encoding a metallo-β-lactamase. Incubation of ceftiofur with either the E. coli transformant or a commercial B. cereus metallo-β-lactamase showed degradation of the drug and formation of the same major metabolite produced by B. cereus P41. These data demonstrate that a metallo-β-lactamase plays a major role in the degradation of ceftiofur by the bovine intestinal bacterium B. cereus P41.

Endometrial tissue and blood plasma concentration of ceftiofur and metabolites following intramuscular administration of ceftiofur crystalline free acid to mares

Systemic administration of ceftiofur crystalline free acid (CCFA) may be a potential treatment for infectious endometritis caused by Streptococcus equi ssp. zooepidemicus (S. zooepidemicus) and other susceptible bacterial organisms in the mare. To determine if i.m. administration of CCFA at the label dose will exceed the minimum inhibitory concentration (MIC) of S. zooepidemicus in the endometrium following single administration and multiple administration protocols. Experimental pharmacokinetic study. Three mares (Group 1) were administered a single i.m. dose of CCFA (6.6 mg/kg bwt) and blood and endometrial biopsies were collected at selected intervals for 144 h. Six additional mares (Groups 2 and 3) received CCFA at times 0, 4, 11 and 18 days, and were sampled at predetermined times for 25 or 49 days, respectively. Plasma and tissue samples were analysed by high-pressure liquid chromatography with tandem mass spectrometry for desfuroylceftiofur acetamide concentration, which is a direct measure of all ceftofur and ceftiofur metabolites in the sample. A mean plasma desfuroylceftiofur acetamide concentration of 0.367 ± 0.0162 μg/ml (mean ± s.e.) was detected at 96 h following administration. Mean endometrial tissue concentration was 0.510 ± 0.0418 μg/g at 96 h and exceeded the MIC for S. zooepidemicus (0.25 μg/ml) throughout the 144 h monitoring period for Group 1. Mares in Groups 2 and 3, given multiple doses of CCFA, maintained plasma concentrations above the MIC for S. zooepidemicus for 25 days. Endometrial tissue levels remained above the MIC at most data collection points for 25 days. Ceftiofur crystalline free acid reaches appropriate endometrial tissue values to exceed the MIC of S. zooepidemicus, a common cause of bacterial endometritis. Therefore, CCFA should be effective in the treatment of equine bacterial endometritis caused by S. zooepidemicus and other susceptible bacterial pathogens in the mare.

PHARMACOKINETICS OF CEFTIOFUR CRYSTALLINE-FREE ACID (EXCEDE STERILE SUSPENSION®) ADMINISTERED VIA INTRAMUSCULAR INJECTION IN WILD CALIFORNIA SEA LIONS (ZALOPHUS CALIFORNIANUS)

The pharmacokinetics of ceftiofur crystalline-free acid (EXCEDE Sterile Suspension, 200 mg ceftiofur equivalents/ml) were determined for the California sea lion (Zalophus californianus). A single dose of EXCEDE was administered intramuscularly at 6.6 mg/kg to 12 wild California sea lions during rehabilitation. The first 10 animals were each assigned to two blood collection time points, with a total of 10 time points at: 6, 12, 24, 48, 72, 96, 120, 144, 168, and 192 hr after administration of the drug. An additional two animals were sampled 1, 2, 3, 4, 5, and 6 hr postinjection. Plasma was separated within 10 min of blood collection and stored at -20 degrees C until analysis. Plasma concentrations of ceftiofur, desfuroylceftiofur, and related metabolites, were determined using liquid chromatography with tandem mass spectrometry (MS). Maximum plasma concentrations of ceftiofur and related metabolites were observed 24 hr postdosing with a mean concentration of 3.6 microg/ml. The half life (60 hr) and area under the curve (270 microg x hr/ml) were also determined. These data indicate that a single dose of EXCEDE at 6.6 mg/kg i.m. would likely maintain a mean plasma drug level >0.6 microg/ml for 5 days and >0.5 microg/ml for 8 days.

Do antibiotic residues in soils play a role in amplification and transmission of antibiotic resistant bacteria in cattle populations?

When we consider factors that contribute to the emergence, amplification, and persistence of antibiotic resistant bacteria, the conventional assumption is that antibiotic use is the primary driver in these processes and that selection occurs primarily in the patient or animal. Evidence suggests that this may not always be the case. Experimental trials show that parenteral administration of a third-generation cephalosporin (ceftiofur) in cattle has limited or short-term effects on the prevalence of ceftiofur-resistant bacteria in the gastrointestinal tract. While this response may be sufficient to explain a pattern of widespread resistance to cephalosporins, approximately two-thirds of ceftiofur metabolites are excreted in the urine raising the possibility that environmental selection plays an important additive role in the amplification and maintenance of antibiotic resistant E. coli on farms. Consequently, we present a rationale for an environmental selection hypothesis whereby excreted antibiotic residues such as ceftiofur are a significant contributor to the proliferation of antibiotic resistant bacteria in food animal systems. We also present a mathematical model of our hypothesized system as a guide for designing experiments to test this hypothesis. If supported for antibiotics such as ceftiofur, then there may be new approaches to combat the proliferation of antibiotic resistance beyond the prudent use mantra.

Determination of ceftiofur derivatives in serum, endometrial tissue, and lochia in puerperal dairy cows with fever or acute puerperal metritis after subcutaneous administration of ceftiofur crystalline free acid

Acute puerperal metritis (APM) is one of the most common diseases during the puerperal period. Systemic administration of ceftiofur for 5 consecutive days has been shown to be effective for treatment of APM. The objective of this study was to determine concentrations of ceftiofur derivatives in serum, endometrial tissue, and lochia of cows with fever postpartum or APM 4 to 6d after treatment with a single subcutaneous dose of 6.6mg of ceftiofur crystalline free acid (CCFA)/kg of estimated BW at the base of the ear. In the first experiment, samples from CCFA-treated cows with fever postpartum or APM (n=42) were taken on d 4, 5, or 6 after treatment. Concentrations of ceftiofur derivatives were quantified using an HPLC assay. Concentrations of active ceftiofur metabolite desfuroylceftiofuracetamide (DCA) were greatest at d 4 after treatment with CCFA in all samples, but they were considerably lower than the concentrations of DCA in healthy postpartum cows treated with the same dose of CCFA. The concentrations of DCA in serum, endometrial tissue, and lochia were affected by odor of vaginal discharge before treatment with CCFA. Mean concentrations of DCA could be detected above the reported minimal drug concentrations (minimum inhibitory concentrations, MIC) required to inhibit relevant pathogens such as Escherichia coli and Arcanobacterium pyogenes in serum on all days and in endometrial tissue and lochia only on d 4 in CCFA-treated cows with fetid vaginal discharge before treatment. In the second experiment, samples from CCFA-treated cows with APM (n=8) were taken on d 0 (before treatment) and d 4, 5, and 6 after treatment. Mean concentrations of DCA in serum and lochia were similar on d 4 to 6 in both laboratories. Furthermore, determined concentrations of DCA from both laboratories were correlated for serum and lochia. Mean concentrations of DCA could be detected above the reported MIC in serum and lochia only on d 4. Our 2 experiments demonstrated that in postpartum cows with fever postpartum or APM concentrations above the MIC for relevant bacteria (>0.5μg/mL or >0.5μg/g) of DCA could be sustained only for 4 (serum: 15/17; endometrial tissue: 2/17; lochia: 1/16) to 5d (serum: 10/13; endometrial tissue: 1/13; lochia: 2/12) after a single treatment with CCFA only in a certain proportion of cows. Overall, our data provide first pharmacological evidence that a single subcutaneous administration of 6.6g of CCFA/kg of BW might not be sufficient to efficaciously treat APM in postpartum dairy cows.

Urine from Treated Cattle Drives Selection for Cephalosporin Resistant Escherichia coli in Soil

The U.S. Food and Drug Administration recently issued new rules for using ceftiofur in food animals in part because of an increasing prevalence of enteric bacteria that are resistant to 3rd-generation cephalosporins. Parenteral ceftiofur treatment, however, has limited effects on enteric bacteria so we tested the hypothesis that excreted ceftiofur metabolites exert significant selection pressure for ceftiofur-resistant Escherichia coli in soil. Test matrices were prepared by mixing soil with bovine feces and adding urine containing ceftiofur metabolites (CFM) (0 ppm, ∼50 ppm and ∼100 ppm). Matrices were incubated at 23°C or 4°C for variable periods of time after which residual CFM was quantified using a bioassay. Bla CMY-2 plasmid-bearing ceftiofur resistant (cefR) E. coli and one-month old calves were used to study the selection effects of CFM and transmission of cefR bacteria from the environment back to animals. Our studies showed that urinary CFM (∼13 ppm final concentration) is biologically degraded in soil within 2.7 days at 23°C, but persists up to 23.3 days at 4°C. Even short-term persistence in soil provides a >1 log10 advantage to resistant E. coli populations, resulting in significantly prolonged persistence of these bacteria in the soil (∼two months). We further show that resistant strains readily colonize calves by contact with contaminated bedding and without antibiotic selection pressure. Ceftiofur metabolites in urine amplify resistant E. coli populations and, if applicable to field conditions, this effect is far more compelling than reported selection in vivo after parenteral administration of ceftiofur. Because ceftiofur degradation is temperature dependent, these compounds may accumulate during colder months and this could further enhance selection as seasonal temperatures increase. If cost-effective engineered solutions can be developed to limit ex vivo selection, this may limit proliferation for ceftiofur resistant enteric bacteria while preserving the ability to use this important antibiotic in food animal production.

A determinative and confirmatory method for ceftiofur metabolite desfuroylceftiofur cysteine disulfide in bovine kidney by LC–MS/MS

Ceftiofur is a cephalosporin β-lactam antibiotic widely used for treating certain bacterial infections in beef and dairy cattle. The regulatory HPLC–UV method for ceftiofur residues in animal tissues is time consuming and non-specific. Additionally, because the regulatory method involves chemical reactions to convert the metabolites into a single moiety, it is virtually impossible to incorporate the procedure into a multi-residue method. Ceftiofur residue violations in beef and dairy cattle have been frequently reported and therefore an improved method is needed. Herein we report a rapid and sensitive LC–MS/MS method for the determination and confirmation of ceftiofur metabolite, desfuroylceftiofur cysteine disulfide (DCCD), in bovine kidney tissue. The new method utilizes a simple extraction with phosphate buffer followed by SPE cleanup. A deuterated internal standard was synthesized and used for quantitation. The matrix-based calibration curve was linear from 25 to 2000ng/g. The average accuracy for control kidney samples from six different sources fortified at 50–1000ng/g was 97.7–100.2% with CV≤10.1%. The limit of confirmation was 50ng/g.

Disposition of desfuroylceftiofur acetamide in serum, placental tissue, fetal fluids, and fetal tissues after administration of ceftiofur crystalline free acid (CCFA) to pony mares with placentitis

The objective of this study was to determine the pharmacokinetics of CCFA in mares with placentitis and evaluate the disposition of the drug in fetal fluids, fetal membranes, colostrum, and serum of foals. A secondary objective was to obtain pilot data regarding the efficacy of CCFA for improving foal survival in mares with placentitis. Twelve pregnant pony mares were enrolled in the study, inoculated with Streptococcus zooepidemicus, intracervically and assigned to one of three groups: CEFT (n = 3; administered CCFA only; 6.6 mg/kg, i.m., q96h); COMBO (n = 6; administered combination therapy of CCFA, altrenogest, and pentoxifylline); UNTREAT (n = 3, no treatment). Treatment was initiated at the onset of clinical signs. Concentrations of desfuroylceftiofur acetamide (DCA), the acetamide derivative of ceftiofur and desfuroylceftiofur metabolites, were measured using high-performance liquid chromatography. Maximum and minimum serum concentrations of DCA at steady state in treated mares were 2.40±0.40 μg/mL and 1.06±0.29 μg/mL, respectively. Concentration of DCA in colostrum was 1.51±0.60 μg/mL. DCA concentrations in placenta and fetal tissues were very low (median = 0.03 μg/mL) and below the minimum inhibitory concentration of relevant pathogens. DCA was not detected in amniotic fluid or foal serum. Treatment did not appear to improve foal survival (CEFT: 0/3; COMBO: 2/6; UNTREAT: 2/3). Bacteria were recovered from the uterus of most mares postpartum and from blood cultures of most foals regardless of treatment.

Assessment of liquid chromatography–tandem mass spectrometry approaches for the analysis of ceftiofur metabolites in poultry muscle

The use of cephalosporin antibiotics in veterinary practice is likely to play an important role in the development of β-lactam-resistant bacteria. To detect off-label cephalosporin antibiotic usage, an analytical method is needed that, besides the native compound, also detects their active metabolites. In this paper, the applicability of three approaches for the quantitative analysis of ceftiofur using LC–MS/MS is assessed, viz. (A) analysis of ceftiofur, desfuroylceftiofur and/or desfuroylceftiofur cystein disulfide, (B) derivatisation of ceftiofur metabolites to desfuroylceftiofur acetamide and (C) chemical hydrolysis using ammonia, to produce a marker compound for ceftiofur. We found that approach A was not suited for quantitative analysis of total ceftiofur concentration or for effectively detecting off-label use of ceftiofur. Approach B resulted in adequate quantitative results, but was considered a single compound method because it depends on cleavage of a thioester group, which is present in only a limited number of cephalosporin antibiotics. Approach C showed adequate quantitative results but, in contrast to approach B, it is applicable to a range of cephalosporin antibiotics. Therefore, it is applicable as a broad quantitative screening of cephalosporin compounds in poultry tissue samples to indicate off-label use of cephalosporins in poultry breeding. Based on this study, it was concluded that approach C is the most suitable to detect off-label use of a range of cephalosporin antibiotics.

Pharmacokinetics of ceftiofur crystalline free acid after single subcutaneous administration in lactating and nonlactating domestic goats (Capra aegagrus hircus)

Six nonlactating and six lactating adult female goats received a single subcutaneous injection of ceftiofur crystalline free acid (CCFA) at a dosage of 6.6 mg/kg. Blood samples were collected from the jugular vein before and at multiple time points after CCFA administration. Milk samples were collected twice daily. Concentrations of ceftiofur and desfuroylceftiofur-related metabolites were measured using high-performance liquid chromatography. Data were analyzed using compartmental and noncompartmental approaches. The pharmacokinetics of CCFA in the domestic goat was best described by a one compartment model. Mean (±SD) pharmacokinetic parameters were as follows for the nonlactating goats: area under the concentration time curve(0-∞) (159 h·μg/mL ± 19), maximum observed serum concentration (2.3 μg/mL ± 1.1), time of maximal observed serum concentration (26.7 h ± 16.5) and terminal elimination half life (36.9 h; harmonic). For the lactating goats, the pharmacokinetic parameters were as follows: area under the concentration time curve(0-∞) (156 h·μg/mL ± 14), maximum observed serum concentration (1.5 μg/mL ± 0.4), time of maximal observed serum concentration (46 h ± 15.9) and terminal elimination half life (37.3 h; harmonic). Ceftiofur and desfuroylceftiofur-related metabolites were only detectable in one milk sample at 36 h following treatment. There were no significant differences in the pharmacokinetic parameter between the nonlactating and lactating goats.

Pharmacokinetics of intravenous ceftiofur sodium and concentration in body fluids of foals

The objectives of this study were to determine pharmacokinetics of intravenous (i.v.) ceftiofur in foals, to compare ultra-high performance liquid chromatography tandem mass spectometry (UPLC-MS/MS) and microbiologic assay for the measurement of ceftiofur concentrations, and to determine the minimum inhibitory concentration (MIC) of ceftiofur against common equine bacterial pathogens. In a cross-over design, ceftiofur sodium was administered i.v. to six foals (1-2 days-of-age and 4-5 weeks-of-age) at dosages of 5 and 10 mg/kg. Subsequently, five doses of ceftiofur were administered i.v. to six additional foals between 1 and 5 days of age at a dose of 5 mg/kg q 12 h. Concentrations of desfuroylceftiofur acetamide (DCA), the acetamide derivative of ceftiofur and desfuroylceftiofur-related metabolites were measured in plasma, synovial fluid, urine, and CSF by use of UPLC-MS/MS. A microbiologic assay was used to measure ceftiofur activity for a subset of plasma samples. Following i.v. administration of ceftiofur at a dose of 5 mg/kg to 1-2 day-old foals, DCA had a t(1/2) of 7.8 +/- 0.1 h, a body clearance of 74.4 +/- 8.4 mL/h/kg, and an apparent volume of distribution of 0.83 +/- 0.09 L/kg. After multiple i.v. doses at 5 mg/kg, DCA concentrations in CSF were significantly lower than concurrent plasma concentrations. Ceftiofur activity using a microbiologic assay significantly underestimated plasma concentrations of DCA. The MIC of ceftiofur required to inhibit growth of 90% of isolates of Escherichia coli, Pasteurella spp, Klebsiella spp, and beta-hemolytic streptococci was <0.5 microg/mL. Intravenous administration of ceftiofur sodium at the rate of 5 mg/kg every 12 h would provide sufficient coverage for the treatment of susceptible bacterial isolates.

Validation Study of a Receptor-Based Lateral Flow Assay for Detection of Beta-Lactam Antibiotics in Milk

A validation study designed to meet the requirements of the AOAC Research Institute and the U.S. Food and Drug Administration (FDA), Center for Veterinary Medicine, was conducted for a receptor-based, immunochromatographic method (BetaStar US) for detection of beta-lactam antibiotic residues in raw, commingled bovine milk. The assay was found to detect amoxicillin, ampicillin, cephapirin, cloxacillin, and penicillin G at levels below the FDA tolerance/safe levels but above the maximum sensitivity thresholds established by the National Conference on Interstate Milk Shipments. Results of the Part I (internal) and Part II (independent laboratory) dose-response studies using spiked samples were in very close agreement for all five drugs tested, with differences between the Part I and Part II 90/95% sensitivity values ranging from 0 to 1 ppb. The test was able to detect all five drugs at the approximate 90/95% sensitivity levels when present as incurred residues in milk collected from cows that had been treated with the specific drug. A sixth drug, ceftiofur, was found to be undetectable at levels of < or = 500 ppb (as total ceftiofur metabolites from incurred residues in milk samples). The selectivity of the assay was 100%, because no false-positive results were obtained in tests of >1000 control milk samples. The assay was found to be applicable to the testing of frozen raw milk samples. Results of ruggedness experiments established the operating parameter tolerances for the BetaStar US assay. Results of cross-reactivity testing established that the assay detects certain other beta-lactam drugs (dicloxacillin and ticarcillin), but it does not cross-react with any of 30 drugs belonging to other classes. Abnormally high bacterial or somatic cell counts in raw milk produced no interference with the ability of the test to detect beta-lactams at tolerance/safe levels.

Penetration of ceftiofur into sterile vs. Mannheimia haemolytica‐infected tissue chambers in beef calves after subcutaneous administration of ceftiofur crystalline free acid sterile suspension in the ear pinna

The effect of Mannheimia haemolytica infection on the penetration of ceftiofur and desfuroylceftiofur metabolites into tissue chambers was studied in cattle after subcutaneous administration of ceftiofur crystalline free acid sterile suspension (CCFA-SS). Four tissue chambers were implanted subcutaneously in each of 12 calves. Approximately 45 days after implantation, two chambers were inoculated with M. haemolytica (10(6) colony-forming units per chamber) while the remaining two chambers were inoculated with sterile phosphate-buffered saline. Twenty-four hours after inoculation, CCFA-SS was administered subcutaneously in the middle third of the caudal ear pinna of each calf. Chamber fluid and blood samples were collected at predetermined times for 10 days following dosing and analyzed for ceftiofur and desfuroylceftiofur metabolites by high-performance liquid chromatography. Concentrations of ceftiofur and desfuroylceftiofur metabolites in plasma and tissue chamber fluid remained above a threshold of 0.2 microg/mL for at least 8 days. Infected tissue chamber fluid concentrations of ceftiofur and desfuroylceftiofur metabolites were significantly higher than those in non-infected tissue chamber fluid, which correlated with significantly higher total protein concentration in infected tissue chambers. These results indicate that single subcutaneous administration of CCFA-SS at 6.6 mg/kg can be expected to provide effective therapy of susceptible bacterial infections for a period of at least 1 week.

Comparison of the use of regulatory assays and high-performance liquid chromatography for detection of residues of ceftiofur sodium metabolites in tissue specimens of culled dairy cattle

To compare the results of regulatory screening and confirmation assays with those of high-performance liquid chromatography (HPLC) in the detection of ceftiofur metabolites in the tissues of culled dairy cattle. 17 lactating Holstein dairy cows. Daily IM injections of ceftiofur sodium were administered at a dose of 2.2 mg of ceftiofur equivalents/kg (n = 6) or 1.0 mg of ceftiofur equivalents/kg (10) for 5 days. Following withdrawal times of 12 hours (high-dose ceftiofur) and either 5 or 10 days (low-dose ceftiofur), cows were slaughtered and liver, kidney, and diaphragmatic muscle specimens were harvested and analyzed by HPLC and standard regulatory methods that included the following assays: the swab test on premises, the fast antimicrobial screen test, the calf antibiotic and sulfa test, and the 7-plate bioassay confirmation test. In all tissue specimens, residues of ceftiofur and desfuroylceftiofur-related metabolites, as measured by HPLC, were less than regulatory tolerance, as defined by the FDA. False-positive screening assay results were more likely for tissue specimens that had been frozen for shipment to a federal laboratory, compared with fresh tissue specimens that were assayed at the slaughter establishment (23% vs 3% false-positive results, respectively). The observation that fresh tissues had negative results on screening assays, whereas subsets of the same tissue specimens had false-positive results on screening assays following freezing, suggests that freezing and thawing interferes with microbial inhibition-based regulatory screening assays.