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Pharmacokinetics of a Single Intramuscular Injection of Long-Acting Ceftiofur Crystalline-Free Acid in Cattle Egrets (Bubulcus ibis).

Abstract: We determined the pharmacokinetic properties of ceftiofur crystalline-free acid (CCFA), a long-acting antibiotic, after a single intramuscular injection in cattle egrets (Bubulcus ibis). A dose of 20 mg/kg was administered intramuscularly to 18 birds and blood samples were collected via jugular venipuncture at 1. 2, 4, 8, 12, 24, 48, 72. 96, 120, 144, 168, 192, 216, and 240 hours after CCFA administration. Plasma concentrations of ceftiofur free acid equivalents (CFAEs) were measured via high-performance liquid chromatography. The minimum inhibitory concentration (MIC) of 1 [micro]g/mL was reached by 1 hour after administration and remained higher than the MIC for at least 72 hours in all birds. This target concentration is effective for many bacterial infections in avian species. The area under the plasma concentration versus time curve was 451.3 [h.sup.*] [micro]g/mL, maximum plasma concentration was 16.22 [micro]g/mL, time to maximum plasma concentration was 3.2 hours, mean harmonic half-life was 37.92 hours, and time that the concentrations of CFAEs were higher than the target MIC was a minimum of 72 hours.

Key words: pharmacokinetic, ceftiofur crystalline-free acid, avian, cattle egret, Bubulcus ibis

Introduction

The cattle egret (Bubulcus ibis), a bird commonly held in zoos and other institutions. It usually is exhibited in large mixed-species flocks where they experience significantly less stress than when housed as individuals or in a small group, which often is necessary for medical treatment. Bacterial infections are a common cause of disease in many avian species, including the egret. (1) Many antibiotics used in avian species require single to multiple times a day dosing to be effective. (2) For many birds, handling multiple times to treat these infections is very stressful. The use of a long-acting injectable antibiotic is very appealing. It allows the clinician to decrease the amount of handling of an individual bird and, therefore, decrease the amount of stress and potential injury they experience.

Ceftiofur crystalline-free acid (CCFA; Excede, Zoetis, Parsippany, NJ, USA) is a long-acting formulation that was developed for use as a single-dose antibiotic in cattle and swine and as a 2-dose antibiotic in horses. It is a third-generation cephalosporin, time-dependent, bactericidal antibiotic. The drug acts by inhibiting bacterial cell wall synthesis. It is efficacious against a variety of gram-positive, gram-negative, and some anaerobic bacteria. (3)

Avian susceptibility studies of minimum inhibitory concentrations (MICs) have been determined for many organisms. (1,4-7) For time-dependent antibiotics, the amount of time that serum concentrations remain above the MIC is the most significant parameter for determining efficacy of the antibiotic. (8) The dose and frequency of dosing varies widely between different avian species. Therefore, CCFA must be evaluated in each target species. (9-12) Using the incorrect dosage can result in antibiotic resistance and/or failure to eliminate the infection. (2)

There currently is no information on the pharmacokinetics of antibiotics in the cattle egret or in any member of the family Ardeidae, which includes egrets, bitterns, and herons. We examined the pharmacokinetics of CCFA after a single intramuscular injection in the cattle egret.

Materials and Methods

Animals

A total of 18 cattle egrets (9 male, 9 female) were included in the primary study, including 6 from the pilot study. All birds were 3 to 4 years of age and weighed between 0.28 and 0.40 kg (mean, 0.34 kg). All birds had been sexed previously by laparoscopy. Birds were housed in the veterinary hospital at Omaha's Henry Doorly Zoo and Aquarium for 2 weeks to allow them to acclimate before the start of the study. Each bird was determined to be in good health based on results of physical examination, CBC and plasma biochemical panel to evaluate their health and to provide blank plasma for the production of standard curves for ceftiofur analysis.

For the pilot study, the birds were weighed and received an intramuscular (IM) injection of CCFA (100 mg/mF) at a dose of 10 or 20 mg/kg IM in the right pectoral muscle administered using a 1-mL syringe and a 22-gauge needle. Six birds were included in each dose group, with 2 birds in each time group. Blood samples were collected from the right jugular vein using a 1-mL syringe and a 23-gauge needle. Samples were collected at 1, 8, 48, 120, and 192 hours for group 1; at 2, 12, 72, 144, and 216 hours for group 2; and at 4, 24, 96, 168, and 240 hours for group 3. Blood was placed in lithium heparin tubes and the plasma was separated via centrifugation. The plasma was transferred to cryogenic storage vials and frozen at -70[degrees]C (-94[degrees] F) within 1 hour of collection until sample analysis.

Based on the results of the pilot study, a dose of 20 mg/kg was used for the primary study. An additional 12 birds were weighed and received an IM injection of CCFA. Four birds, 2 males and 2 females, were in each of 3 time groups. Blood samples were collected at the same times as for the pilot study and processed similarly. This resulted in a total of six birds at each time period using the data from the pilot and primary studies. The primary study was completed 5 months after the pilot study.

Plasma analysis

Ceftiofur in plasma samples was analyzed using reversed phase high performance liquid chromatography (HPFC). The system consisted of a 2695 separations module and a 2487 ultraviolet detector (Waters, Milford, MA, USA). Separation was attained on a Symmetry [C.sub.18] 4.6 X 250 mm (5 [micro]m) with a 5-pm Symmetry guard column (Waters). The mobile phase was a mixture of: (solution A) 0.1% trifluroracetic acid (TFA) in water and (solution B) 0.1% TFA in acetonitrile. The mixture was pumped at a starting gradient of 90% solution A and 10% solution B and was adjusted to 75% solution A and 25% solution B over 25 minutes, and back to initial conditions over 3 minutes. The drug was quantified using ultraviolet (UV) detection at 265 nm with a flow rate of 1.0 mF/min at ambient temperature, which is 23[degrees]C (73.4[degrees]F).

Ceftiofur was extracted from plasma samples by a slightly modified derivitization method that converts ceftiofur and all desfuroylceftiofur metabolites to desfuroylceftiofur acetamide. (13) Briefly, previously frozen plasma samples were thawed and vortexed, and 100 [micro]L were transferred to a clean test tube; then, 15 [micro]L internal standard (100 [micro]g/mL cefotaxime) were added. Then, 7 mL 0.4% dithioerythritol in borate buffer was added and the tubes were placed in a 50[degrees]C (112[degrees]F) water bath for 15 minutes. The tubes were removed and allowed to cool to room temperature. Then, 1.5 mL iodoacetaminde buffer was added. The solution then was passed through a prewet Oasis HFB extraction column (Waters). Samples were eluted with a 5% glacial acetic acid in methanol solution, which then was evaporated to dryness under a steady stream of nitrogen gas. Samples were reconstituted in 200 [micro]L mobile phase and 50 [micro]L were injected into the HPFC system.

Standard curves for plasma analysis were prepared by spiking untreated plasma with ceftiofur, which produced a linear concentration range of 0.1 to 100 [micro]g/mF. Spiked standards were treated exactly as plasma samples. Average recovery was 99% for ceftiofur. Intra-assay variability ranged from 0.7% to 4.5% and interassay variability ranged from 3.6% to 8.8% for ceftiofur, respectively. The lower limit of quantification was 0.1 [micro]g/ mL.

Pharmacokinetic analysis

Plasma concentrations of ceftiofur free acid equivalents (CFAEs) were analyzed by means of noncompartmental analysis with Phoenix, WinNonlin (Certra Menlo Park, CA, USA). Means and standard deviations (SDs) were calculated for the elimination rate constant ([[lambda].sub.z]), time to maximum plasma concentration ([T.sub.max]), maximum observed plasma concentration ([C.sub.max]), area under the plasma concentration time curve ([AUC.sub.0-[infinity]]), and mean transit time ([MTT.sub.0-[infinity]]). Mean transit time and AUC were calculated from time 0 to infinity. The harmonic mean and pseudo-SD were calculated for terminal half-life.

Results

Animals

All birds in the pilot and primary studies were healthy on results of the physical examination, CBC, and plasma biochemical analyses. No adverse drug effects, including injection site reactions, were observed in any bird. All birds were tolerant of the handling and blood collection. All birds were returned to the larger flock 2 weeks after completion of the study, and no adverse effects were observed subjectively based on observations of normal behavior, appetite, and fecal production.

Pilot study

In the birds given 10 mg/kg CCFA, ceftiofur equivalents were detected at 1 hour and peaked between 2 and 4 hours. The concentrations were below 1.0 [micro]g/mL after 48 hours and no ceftiofur equivalents were detectable after 192 hours. The data from the birds given 20 mg/kg are included in the primary study results since drug administration, sample collection, and plasma analysis were identical.

Primary study

Ceftiofur free acid equivalents were detected from 1 through 240 hours. The mean ceftiofur equivalent concentration versus time profile is illustrated in Figure 1. Plasma concentrations of CFAEs remained above 1 pg/mL in all birds at 72 hours and in 50% of birds at 96 hours. Mean pharmacokinetic parameters were determined (Table 1).

Discussion

Excede is a long-acting, third-generation cephalosporin with broad-spectrum antimicrobial activity. It has been shown to provide a therapeutic level for target bacteria starting within hours and lasting for at least 6 days in cattle, 8 days in swine, 10 days in horses after two doses, (3) 5 days in California sea lions (Zalophus californianus), (14) 7 to 10 days in Asian elephants (Elephas maxim us), (15) 12 days in bearded dragons (Pogona vitticeps), (16) 5 days in ball pythons (Python regius), (17) and 7 days in the male rhesus macaque (Macaca mulatta). (18) However, many of these studies used different doses and evaluated different [MIC.sub.90]s. The studies from avian species are summarized in Table 2. Great variation in drug duration is obvious between species, and the longevity of this antibiotic must be determined in more avian species.

The reported [MIC.sub.90] of ceftiofur for numerous avian bacterial pathogens is [less than or equal to] 1 [micro]g/mL. This includes Enterobacter species, Pasteurella spp, Staphylococcus aureus, (9) Proteus species, Klebsiella species, (4,9) Salmonella species, and Escherichia coli, (4,5,7) Salmonella species, E. coli, staphylococci and Pasteurella species are some of the causative agents of septicemia and airsacculitis in ducks. (7) Salmonella enterica has been found in infected wild cattle egrets and may pose a health risk to humans living in close proximity. (19)

American black ducks (Anas rubripes) administered CCFA IM had plasma concentrations [greater than or equal to] 1 [micro]g/ mL for a longer period than those dosed subcutaneously (SC), and CCFA seemed to have greater bioavailability after IM than subcutaneous administration although this was not fully tested via statistical analysis. (9) In the American flamingo (Phoenicopterus ruber), values were less consistent with the SC than with the IM route. (10) We chose only to use the IM route in this study based on those results.

In our pilot study, 10 mg/kg CCFA produced ceftiofur equivalent levels [less than or equal to] 1 [micro]g/mL for 48 hours. A total of 20 mg/kg in our primary study allowed for an additional 24 hours at a therapeutic level. In previous CCFA studies, the avian species that is closest to the cattle egret in size is the ringneck dove (Streptopelia risoria), approximately one-half the size of the cattle egret. A dose of 50 mg/kg was needed to obtain a therapeutic level for 96 hours in the dove compared to 20 mg/kg for 72 hours in the egret. (20) The red-tailed hawk (Buteo jamaicensis) is approximately 4 times the size of the egret, yet the same dose only achieved therapeutic levels for an additional 24 hours in the hawk. (11) Obviously, the average weight of a species cannot be used to determine the dose or duration of CCFA. Without additional studies, the maximum amount of drug that could be injected safely into the pectoral muscle of the cattle egret is impossible to determine. If a higher dose was used potentially to obtain a longer therapeutic level and, therefore, decrease the amount of handling of an infected individual, the more concentrated cattle CCFA formulation of 200 mg/mL could be used.

The mean [T.sub.max] for the cattle egret (3.2 hours, 20 mg/kg) was much shorter than that for the helmeted guineafowl (Numida meleagris; 19.3 hours, 10 mg/kg), (12) the American flamingo (27 hours, 10 mg/kg), (10) and the American black duck (24 hours, 10 mg/kg). (9) It was most similar to the red-tailed hawk (6.6 hours, 20 mg/kg). (11) The [C.sub.max] was highest in the cattle egret (16.22 [micro]g/mL) followed most closely by the red-tailed hawk (15.1 [micro]g/mL, 20 mg/kg) (11) and then the American black duck (13.07 [micro]g/[micro]L, 10 mg/kg). (9) The American flamingo (7.49 [micro]g/mL, 10 mg/kg) (10) and the helmeted guineafowl (5.26 [micro]g/mL, 10 mg/kg) (12) were both much lower. The harmonic mean for the cattle egret (37.92 hours, 20 mg/kg) is longer than that for the helmeted guineafowl (29 hours, 10 mg/ kg) (12) and the American black duck (32 hours, 10 mg/kg), (9) and shorter than that for the American flamingo (39.9 hours, 10 mg/kg) (10) and red-tailed hawk (50 hours, 20 mg/kg). (11) All bird species from the CCFA studies are in different orders of birds. Physiologic differences between orders and species of birds may help explain the differences in results, as different species would be likely to have different rates of absorption and elimination.

Based on our results, a dose of 20 mg/kg IM in the cattle egret will result in blood ceftiofur equivalent levels above 1 [micro]g/mL for a minimum of 72 hours. Clinical use should be based on culture and sensitivity results. Additional multidose studies, intravenous studies, and studies of higher doses are needed to evaluate fully the pharmacokinetics of CCFA in the cattle egret. Comparison of the pharmacokinetics of the cattle egret to a heron or other egret species would be interesting.

Acknowledgments: We thank the Omaha's Henry Doorly Zoo and Aquarium for their support of this study, the bird department and veterinary hospital staff for their care of the birds, and Joan Swinford, LVT for her assistance with sample processing.

References

(1.) Gerlach H. Bacteria. In: Ritchie BW. Harrison GJ. and Harrison LR, eds. Avian Medicine: Principles and Application. Lake Worth, FL: Wingers; 1994: 949-983.

(2.) Flammer K. Antibiotic drug selection in companion birds. J Exot Pet Med. 2006; 15(3): 166-176.

(3.) Excede [package insert]. New York: Pharmacia & Upjohn Co, Division of Pfizer, Inc, 2011.

(4.) Salmon SA, Watts JL, Yancey RJ. In vitro activity of ceftiofur and its primary metabolite, desfuroylceftiofur, against organisms of veterinary importance. J Vet Diagn Invest. 1996;8(3):332 336.

(5.) Salmon SA, Watts JL. Minimum inhibitory concentration determinations for various antimicrobial aganets against 1570 bacterial isolates from turkey poults. Avian Dis. 2000;44(1):85 98.

(6.) Tell L, Harrenstein L, Wetzlich S, et al. Pharmacokinetics of ceftiofur sodium in exotic and domestic avian species. J Vet Pharmacol Ther. 1998;21 (2):85-91.

(7.) Watts JL, Salmon SA, Yancey RJ, et al. Minimum inhibitory concentrations of bacteria isolated from septicemia and airsacculitis in ducks. J Vet Diagn Invest. 1993;5(4):625 628.

(8.) Vogelman B, Gudmundsson S, Leggett J, et al. Correlation of antimicrobial pharmacokinetic parameters with therapeutic efficacy in an animal model. J Infect Dis. 1988; 158(4):831 847.

(9.) Hope KL, Tell LA, Byrne BA, et al. Pharmacokinetics of a single intramuscular injection of ceftiofur crystalline-free acid in American black ducks (Anas rubripes). Am J Vet Res. 2012;73(5):620-627.

(10.) Kilburn JJ, Cox SK, Backues KA. Pharmocokinetics of ceftiofur crystalline free acid, a long-acting cephalosporin, in American flamingos (Phoenicopterus ruber). J Zoo Wildl Med. 2016;47(2):457-462.

(11.) Sadar MJ, Hawkins MG, Byrne BA, et al. Pharmacokinetics of a single intramuscular injection of ceftiofur crystalline-free acid in red-tailed hawks (Buteo jamaicensis). Am J Vet Res. 2015; 76(12): 1077-1084.

(12.) Wojick KB, Langan JN, Adkesson MJ, et al. Pharmacokinetics of long-acting ceftiofur crystalline-free acid in helmeted guineafowl (Numida meleagris) after a single intramuscular injection. Am J Vet Res. 2011;72(11): 1514-1518.

(13.) De Baere S, Pille F, Croubels S, et al. Highperformance liquid chromatographic-UV detection analysis of ceftiofur and its active metabolite desfuroylceftiofur in horse plasma and synovial fluid after regional intravenous perfusion and systemic intravenous injection of ceftiofur sodium. Anal Chim Acta. 2004;512:75-84.

(14.) Meegan J. Collard WT. Grover GS. et al. Pharmacokinetics of ceftiofur crystalline-free acid (Excede sterile suspension[R]) administered via intramuscular injection in wild California sea lions (Zaloplius californianus). J Zoo Wildl Med. 2013 ;44(3): 714-720.

(15.) Adkesson MJ, Junge RE, Allender MC, et al. Pharmacokinetics of a long-acting ceftiofur crystalline-free acid formulation in Asian elephants (Elephas maximus). Am J Vet Res. 2012;73(10): 1512-1518.

(16.) Churgin SM, Musgrave KE, Cox SK. Sladky KK. Pharmacokinetics of subcutaneous versus intramuscular administration of ceftiofur crystalline-free acid to bearded dragons (Pogona vitticeps). Am J Vet Res. 2014;75(5):453-459.

(17.) Adkesson MJ, Fernandez-Varon E, Cox S, Martin-Jimenez T. Pharmacokinetics of a long-acting ceftiofur formulation (ceftiofur crystalline-free acid) in the ball python (Python regius). J Zoo Wildl Med. 2011;42(3):444 450.

(18.) Salyards GW, Knych HK, Hill AE, et al. Pharmacokinetics of ceftiofur crystalline free acid in male rhesus macaques (Macaca mulatto) after subcutaneous administration. J Am Assoc Lab Anim Sci. 2015;54(5):557 563.

(19.) Phalen DN, Drew ML, Simpson B, et al. Salmonella enterica subsp. enterica in cattle egret (Bubulcus ibis) chicks from central Texas: prevalence, serotypes, pathogenicity, and epizootic potential. J Wildl Dis. 2010;46(2):379-389.

(20.) Valitutto MT, Newton AL, Raphael BL, et al. Pharmacokinetics of a long-acting formulation of ceftiofur (ceftiofur crystalline-free acid) administered intramuscularly in the ringneck dove (Streptopelia risoria). Proc Annu Conf Am Assoc Zoo Vet. 2010:223-224.

Jennifer A. Waldoch, DVM, Sherry K. Cox, PhD, and Douglas L. Armstrong, DVM

From Omaha's Henry Doorly Zoo and Aquarium. 3701 S 10th Street, Omaha. NE 68107, USA (Waldoch. Armstrong) and the University of Tennessee College of Veterinary Medicine. Department of Biomedical and Diagnostic Sciences. 2407 River Drive, Knoxville, TN 37996. USA (Cox).

Caption: Figure 1. Mean [+ or -] SD of plasma ceftiofur free acid equivalent concentration after administration of 20 mg/kg IM of ceftiofur crystalline-free acid in 18 cattle egrets.
Table 1. Pharmacokinetic parameters of ceftiofur
equivalents after administration of 20 mg/kg IM
ceftiofur crystalline-free acid in 18 cattle egrets.

Pharmacokinetic parameter      Value [+ or -] SD

Harmonic mean (h)             37.92 [+ or -] 7.49
Elimination rate constant    0.0183 [+ or -] .0036
  [[lambda].sub.z] (1/h)
[T.sub.max] (h)                3.20 [+ or -] 2.6
[C.sub.max] ([micro]g/mL)     16.22 [+ or -] 5.11
[AUC.sub.0-[infinity]]       451.30 [+ or -] 141.0
  ([h.sup.*][micro]g/mL)
[MRT.sub.0-[infinity]] (h)    47.90 [+ or -] 8.5

Abbreviations: SD, standard deviation; Tmax, time to maximum
plasma concentration; Cmax, maximum observed plasma
concentration; [AUC.sub.0-[infinity]], area under the plasma
concentration time curve from zero to infinity;
[MRT.sub.0-[infinity]], mean transit time from zero to infinity.

Table 2. Summary of studies evaluating pharmacokinetics of ceftiofur
crystalline-free acid in avian species.

                    Average weight   Dose,       Route of
Species              or range, kg    mg/kg    administration

American black           1.14          10           IM
  ducks (9)
American                 >2.4          10           IM
  flamingos (10)
Cattle egret             0.34          20           IM

Helmeted              1.37-1.96        10           IM
  guineafowl (12)
Red-tailed             1.1-1.6         10           IM
  hawks (11)
Red-tailed             1.1-1.6         20           IM
  hawks (11)
Ringneck                 0.17          50           IM
  dove (20)

                          [MIC.sub.90]          Minimal time
Species                       used                 above
                                                [MIC.sub.90],
                                                      h

American black        4 [micro]g/mL (a)             73.3
  ducks (9)
American            [less than or equal to]1         72
  flamingos (10)      [micro]g/mL
Cattle egret        [less than or equal to]1         72
                      [micro]g/mL
Helmeted            [less than or equal to]1         56
  guineafowl (12)     [micro]g/mL
Red-tailed            4 [micro]g/mL (a)              36
  hawks (11)
Red-tailed            4 [micro]g/mL (a)              96
  hawks (11)
Ringneck            [less than or equal to]1         96
  dove (20)           [micro]g/mL

Abbreviation: [MIC.sub.90], minimum inhibitory concentration that
inhibits 90% of the isolates.

(a) The study used a target plasma concentration of 4 [micro]g/mL.
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Title Annotation:Original Study
Author:Waldoch, Jennifer A.; Cox, Sherry K.; Armstrong, Douglas L.
Publication:Journal of Avian Medicine and Surgery
Date:Dec 1, 2017
Words:3486
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