Cefquinome is a fourth-generation cephalosporin developed solely for veterinary use.1 The drug has potent activity against a broad spectrum of bacterial species, such as Riemerella anatipestifer,2 which is a gram-negative bacterium that can cause severe, contagious disease in various avian species.3 Although the pharmacokinetics of cefquinome has been investigated in various animal species, only 1 study4 has been reported regarding the pharmacokinetics in young waterfowl specifically, and the Muscovy ducks (Cairina moschata) in that study were 2 months old. Nevertheless, anatipestifer disease primarily affects young (2- to 8-week-old) goslings and ducklings, with high mortality rates possible.5 Therefore, the purpose of the study reported here was to investigate the pharmacokinetics and bioavailability of cefquinome in goslings and ducklings after IV or IM administration of a single dose and determine whether differences existed in the disposition of 2 formulations—injectable cefquinome sulfate and injectable cefquinome sulfate suspension (a slow-release product)—between goslings and ducklings.
Materials and Methods
Animals
A total of 216 healthy Muscovy ducklings and 216 healthy Sichuan white goslings (Anser cygnoides) were used in the study. All birds were 1 week old and weighed approximately 100 to 120 g. All ducklings and goslings were provided by the Department of Laboratory Animals, Rongchang campus, Southwest University.
Birds were maintained in cages in an environmentally controlled room and fed an antimicrobial-free balanced diet ad libitum with free access to fresh water. They were allowed to acclimate to this setting for 1 week before the experiment began. Animal care and use for the study was in accordance with China's legal requirements for humane treatment, and the study protocol was approved by the Southwest University Animal Care Committee.
Experimental design
Ducklings and goslings were each randomly allocated to 3 treatment groups (n = 72 ducklings or goslings/group) by means of a randomized block design. Each group received a 2-mg/kg dose of cefquinome sulfate; however, ducklings or goslings assigned to group 1 received injectable cefquinome sulfatea I V, those assigned to group 2 received injectable cefquinome sulfatea IM, and those assigned to group 3 received injectable cefquinome sulfate suspensionb IM, in accordance with the manufacturer's instructions.
Jugular venous blood samples were collected from 6 ducklings or goslings (ie, sample collection from each bird was performed only once) at specific time points (group 1: 5, 10, 15, 30, and 45 minutes [0.083, 0.167, 0.25, 0.5, and 0.75 hours, respectively] and 1, 2, 3, 4, 6, 8, and 10 hours after drug administration; group 2: 5, 10, 15, 20, 30, and 45 minutes [0.083, 0.167, 0.25, 0.333, 0.5, and 0.75 hours, respectively] and 1, 2, 4, 6, 8, and 12 hours after drug administration; and group 3: 5, 10, 15, 20, and 30 minutes [0.083, 0.167, 0.25, 0.333, and 0.5 hours, respectively] and 1, 2, 4, 6, 8, 12, and 24 hours after drug administration). Within 3 hours after collection, the samples were centrifuged at 3,000 × g for 5 minutes at 4°C, and harvested plasma was preserved at −20°C.
Analytic method
A modified HPLC method was used to determine plasma cefquinome concentrations.4 The HPLC systemc was equipped with a quaternary pump, online degasser, autosampler, column heater, and UV detector. First, 600 μL of methanold was added to a 300-μL plasma sample by vortex blending for 1 minute, followed by centrifugation at 16,600 × g for 15 minutes at 4°C. Then, the supernatant was transferred to a new tube, dried with nitrogen gas, reconstituted in 300 μL of mobile phase, and filtered through a 0.45-μm nylon syringe filter. The mobile phase consisted of sodium perchloratee-phosphatef-triethylaminee buffer (pH, 3.6) and acetonitriled at a ratio of 88:12 (vol/vol) provided in isocratic form at a flow rate of 1.0 mL/min. Chromatographic separation of cefquinome was achieved on a reverse-phase 250 × 4.6-mm C18 columng with an injection volume of 20 μL. Column temperature was maintained at 30°C. The wavelength for UV detection was 266 nm.
Method validation—Standard working solutions of cefquinome at concentrations of 25, 5, 2.5, 0.5, 0.25, and 0.05 μg/mL were prepared by diluting the cefquinome sulfate stock solutionh (1 mg/mL) with the mobile phase. Then 60 μL of each standard working solution was added to 240 μL of blank duckling and gosling plasma to yield plasma samples spiked with known cefquinome concentrations (5, 1, 0.5, 0.1, 0.05, and 0.01 μg/mL). Cefquinome concentrations were measured 5 times in each spiked sample with the described HPLC method, and the means of the 5 values were used to plot standard curves (peak area vs cefquinome concentration).4 The linear regression equations based on the results for spiked plasma samples from ducklings and goslings were y = 0.4214x + 0.0013 (R2 = 0.9999) and y = 0.3883x + 0.0191 (R2 = 0.9995), respectively.
Percentage recovery and intraday and interday coefficients of variability were determined via repetitive analysis of the plasma samples spiked with cefquinome at concentrations of 0.05, 0.5, and 5 μg/mL (Supplementary Appendix S1, available at: avmajournals.avma.org/doi/suppl/10.2460/ajvr.81.11.873). The lower limits of detection and quantification were 0.01 and 0.03 μg/mL, respectively, and were calculated on the basis of signal-to-noise ratios of 3 and 10, respectively.6 In addition, the HPLC method had an optimal specificity for determining cefquinome by chromatographic analysis, as indicated by the lack of a chromatographic peak for blank plasma samples at the retention times of cefquinome (11.5 to 12.0 minutes; Supplementary Figure S1, available at: avmajournals.avma.org/doi/suppl/10.2460/ajvr.81.11.873).
Pharmacokinetic analysis
Nonlinear least squares regression analysis and computer softwarei were used to fit the plasma concentration-versus-time data to a series of pharmacokinetic models with plasma cefquinome concentration data weighted by 1, 1/c, and 1/c,2 where c is the plasma cefquinome concentration.4 The optimal model was determined by consideration of the values for the weighted residual sums of squares and Akaike information criterion.7 Mean cefquinome concentrations in the 6 plasma samples at each time point were used for calculation of kinetic disposition.
where t is time, A is the zero-time intercept of the distribution slope in the compartment model, B is theconcentration (t) = Ae−α•t + Be−β•t
zero-time intercept of the decrease in plasma concentration of the drug, α is the distribution rate constant, and β is the elimination rate constant.
Values for maximum plasma concentration and tmax were obtained from the fitted concentration-versus-time curves based on results of the compartmental models.8 The bioavailability of cefquinome was calculated as (AUCIM/DoseIM) × 100/(AUCIV/DoseIV).
Results
No adverse effects or signs of intolerance were observed in any duckling or gosling during the entire experiment. Plasma cefquinome concentration-versus-time curves were plotted (Figure 1). Mean plasma cefquinome concentrations in both ducklings and goslings were less than the lower limit of detection of the HPLC assay by 10 hours after IV administration of injectable cefquinome sulfate and by 12 hours after IM administration of injectable cefquinome sulfate (Supplementary Table S1, available at: avmajournals.avma.org/doi/suppl/10.2460/ajvr.81.11.873). However, persistence of detectable cefquinome in plasma samples was noted 24 hours after IM administration of injectable cefquinome sulfate suspension. Pharmacokinetic values derived from compartmental analysis were summarized (Table 1).
Mean ± SD plasma cefquinome concentrations versus time in 1-week-old Muscovy ducklings (Cairina moschata; A) and Sichuan white goslings (Anser cygnoides; B) after administration of a single dose of cefquinome sulfate (2 mg/kg) at 0 hours (n = 6 birds/data point). Birds assigned to group 1 received injectable cefquinome sulfate IV (circles), those assigned to group 2 received injectable cefquinome sulfate IM (squares), and those assigned to group 3 received injectable cefquinome sulfate suspension IM (triangles; n = 72 ducklings or goslings/group). Note that the scales of both axes in each panel are nonlinear. The horizontal dashed line represents the reported MIC of cefquinome for Riemerella anatipestifer wild-type HN68.2
Citation: American Journal of Veterinary Research 81, 11; 10.2460/ajvr.81.11.873
Mean values of pharmacokinetic parameters for cefquinome sulfate after IV or IM administration of a single dose (2 mg/kg) to 1-week-old Muscovy ducklings (Cairina moschata) and Sichuan white goslings (Anser cygnoides).
| Duckling group No.* | Gosling group No.* | |||||
|---|---|---|---|---|---|---|
| Parameter | 1 | 2 | 3 | 1 | 2 | 3 |
| Vdss (L/kg) | 0.042 | NA | NA | 0.432 | NA | NA |
| t1/2α (h) | 0.019 | 0.343 | 0.461 | 0.446 | 0.483 | 0.642 |
| t1/2β (h) | 0.972 | 1.717 | 1.895 | 1.737 | 1.403 | 6.917 |
| tmax (h) | NA | 0.163 | 0.143 | NA | 0.203 | 0.253 |
| Cmax (μg/mL) | NA | 4.010 | 5.721 | NA | 3.400 | 3.431 |
| AUC (μg•h/mL) | 6.248 | 4.220 | 6.047 | 4.396 | 5.008 | 5.411 |
| ClB (L/kg•h) | 0.320 | 0.474 | 0.331 | 0.455 | 0.399 | 0.370 |
| F (%) | NA | 67.5 | 96.8 | NA | 113.9 | 123.1 |
Birds assigned to group 1 received injectable cefquinome sulfate IV, those assigned to group 2 received injectable cefquinome sulfate IM, and those assigned to group 3 received injectable cefquinome sulfate suspension IM (n = 72 ducklings or goslings/group).
ClB = Total body clearance. Cmax = Maximum observed concentration. F = Bioavailability. NA = Not applicable. Vdss = Volume of distribution at steady state.
Discussion
After IV and IM administration of cefquinome (2 mg/kg) in healthy 1-week-old ducklings and goslings, the disposition kinetics of cefquinome were best fitted by a 2-compartment model as reported for chickens (2 mg/kg)1 and 2-month-old ducks (5 mg/kg).4 Mean t1/2β following IV administration was slightly shorter in the ducklings (0.972 hours) than in the goslings (1.737 hours) of the present study and was also shorter than the mean t1/2β reported for chickens (1.29 hours),1 2-month-old ducks (1.57 hours),4 and black swans (1.69 hours).9 Considered together, these findings suggest that cefquinome is rapidly eliminated in waterfowl, particularly in ducklings. For the ducklings of the present study, mean volume of cefquinome distribution at steady state (0.042 L/kg) was considerably lower than that in goslings (0.432 L/kg). This limited distribution could be explained by the low fat solubility and protein-binding activity of cefquinome,10 which may be more pronounced in ducklings.
With IM administration of injectable cefquinome sulfate in ducklings, mean tmax and t1/2α were 0.163 and 0.343 hours, respectively, and were shorter than respective values for goslings in the present study (0.203 and 0.483 hours), chickens (0.25 and 0.58 hours),1 and 2-month-old ducks (0.38 and 0.46 hours).4 These findings indicate rapid absorption and distribution of cefquinome following IM administration, particularly in ducklings. Mean t1/2β in ducklings (1.717 hours) was similar to that of goslings in the present study (1.403 hours), 2-month-old ducks (1.79 hours),4 and chickens (1.35 hours).1 Bioavailability (mean, 113.9%) was excellent in the goslings but was low in the ducklings (67.5%). In goslings, mean bioavailability of both injectable cefquinome sulfate and injectable cefquinome sulfate suspension (123.1%) exceeded 100%, which might be explained by the inclusion of only 1 blood sample/bird in parameter estimations; however, the ducklings, for which bioavailability was < 100%, were sampled similarly. The reason for the higher bioavailability in goslings requires further investigation.
The injectable cefquinome sulfate suspension evaluated in the present study was formulated for slow, sustained release of the drug. Nevertheless, it was rapidly absorbed and distributed after IM administration in ducklings and goslings, as indicated by fast tmax and t1/2α in ducklings (0.143 and 0.461 hours, respectively) and goslings (0.253 and 0.642 hours). These values were compatible with previously reported values for chickens and ducks.1,4 In addition, the bioavailability of injectable cefquinome sulfate suspension in ducklings and goslings was 96.8% and 123.1%, respectively, thereby indicating almost complete absorption. Notably, the t1/2β of injectable cefquinome sulfate suspension in goslings (6.917 hours) was longer than that for injectable cefquinome sulfate (1.403 hours) and also than that for IM cefquinome administration in chickens (1.35 hours)1 and 2-month-old ducks.4 However, this long-lasting effect was not found in ducklings, possibly owing to differences between species in liver microsomal enzyme function or injection-site absorption.
Although cefquinome has not been widely used in waterfowl, the susceptibility of some bacteria of waterfowl origin to cefquinome has been reported. The MIC of cefquinome against R anatipestifer wild-type HN68 is 0.03125 μg/mL.2 To provide a therapeutic effect, many cephalosporins are recommended to be administered at a frequency predictive of efficacy (ie, the time with a serum concentration above the MIC should exceed 40% of the dosing interval).11 Therefore, we would recommend repeated (twice-daily) IM administration of injectable cefquinome sulfate or injectable cefquinome sulfate suspension at 2 mg/kg when treating ducklings with anatipestifer disease. For goslings, once-daily IM administration of injectable cefquinome sulfate suspension and twice-daily IM administration of injectable cefquinome sulfate at 2 mg/kg may provide a plasma cefquinome concentration greater than the MIC for R anatipestifer.
Acknowledgments
Funded by grants from the Fundamental Research Funds for the Central Universities (grant No. XDJK2019B040) and the Chongqing Basic Research and Frontier Exploration project (grant Nos. cstc2018jcyjAX0466 and cstc2017jcyjAX0040).
The authors declare that there were no conflicts of interest.
ABBREVIATIONS
| AUC | Area under the concentration-versus-time curve |
| HPLC | High-performance liquid chromatography |
| MIC | Minimum inhibitory concentration |
| t1/2α | Distribution half-life |
| t1/2β | Elimination half-life |
| tmax | Time to maximum plasma concentration |
Footnotes
Injectable cefquinome (2.5%), Qilu Animal Health Products Co, Jinan, Shandong, China.
Cefquinome suspension injection (2.5%), Qilu Animal Health Products Co, Jinan, Shandong, China.
UltiMate3000, Thermo Fisher Scientific, Waltham, Mass.
Tedia, Fairfield, Ohio.
Kelon Chemical Reagent Factory, Chengdu, China.
Chuandong Chemical Group Co, Chongqing, China.
Neptune C18 column (5 μm), Guangzhou FLM Scientific Instrument Co, Guangzhou, Guangdong, China.
Cefquinome standard (81.2% purity), China Institute of Veterinary Drug Control, Beijing, China.
3P97 practical pharmacokinetics program, Chinese Pharmacological Association, Beijing, China.
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