Red-tailed hawks (Buteo jamaicensis) are one of the most common raptorial species in the United States. Large numbers of these birds are rehabilitated in centers around the country prior to their release back into the wild. In addition, red-tailed hawks are also maintained as nonreleasable animals in educational facilities, as display birds in zoos, and as companions in the falconry community. They are often evaluated for traumatic injuries, some of which include underlying infectious disease processes that require treatment with antimicrobials.
Because red-tailed hawks are at risk for infection with gram-positive and gram-negative organisms, an ongoing need exists for antimicrobials with broad-spectrum activity that can be administered infrequently to minimize distress to birds during handling.1 The physical restraint required to administer antimicrobials to ill raptors can create a great deal of distress for these patients. Few data are available on effective doses and administration frequency of antimicrobials for red-tailed hawks and include only a small number of medications and routes of administration.2–5 To complicate matters further, all of the antimicrobials evaluated in this species require administration at least once per day to be effective.
When a bird will accept food, antimicrobials can be administered through the food; however, ill birds may not accept food or may require frequent dose administration. Availability of an injectable antimicrobial that requires less frequent administration would considerably decrease the amount of handling associated with antimicrobial administration.
Ceftiofur is a third-generation cephalosporin antimicrobial with activity against various gram-positive and gram-negative, aerobic and anaerobic bacteria encountered by domestic animals.6 Like other cephalosporins, ceftiofur inhibits bacteria cell wall synthesis; it is predominately bactericidal and is considered time dependent in that the antibiotic should remain at the binding site for a sufficient period for the metabolic processes of the bacteria to be sufficiently inhibited. Several formulations of ceftiofur are commercially available; however, only 2 have been evaluated in birds. To the authors’ knowledge, no ceftiofur formulation has been evaluated in any raptorial species. The first formulation evaluated in other avian species was ceftiofur sodium, which allows once-daily administration in mammals.7 However, for cockatiels, orange-winged Amazon parrots, turkey poults, and chicken chicks, ceftiofur sodium requires administration multiple times per day.7
A newer injectable formulation, CCFA, is a sustained-release cephalosporin approved by the US FDA for treatment of gram-positive and gram-negative, aerobic and anaerobic bacterial infections of cattle and swine and of Streptococcus equi subsp zooepidemicus infections of horses.8–10 Because of its bactericidal, broad-spectrum, and long-acting properties, CCFA has been suggested as a potentially useful antimicrobial treatment for birds. To the authors’ knowledge, only 2 reports6,11 have been published on use of this antimicrobial in avian species. The pharmacokinetics of CCFA has been evaluated in American black ducks (Anas rubripes)6 and helmeted guineafowl (Numida meleagris)11 at a dose of 10 mg/kg, IM. The suggestion arising from those studies was that CCFA be administered IM every 72 hours to reach a TPC of 4.0 μg/mL in black ducks6 and a plasma concentration suitable to treat guineafowl with bacterial infections for which the MIC of ceftiofur is ≤ 1.0 μg/mL.11
In addition to determining the dose and administration frequency of an antimicrobial, it is also important to determine whether any adverse effects are associated with its administration. In the studies6,11 of CCFA in black ducks and guineafowl, only visual evaluations of the injection sites were performed. Reports1,12 of another injectable antimicrobial, enrofloxacin, as well as anecdotal reports from clinicians treating raptorial species with CCFA at doses higher than 10 mg/kg have indicated that muscle necrosis occurs with CCFA administration, even with a single dose; whether the same occurs in red-tailed hawks needs to be determined. The potential for muscle necrosis is particularly important for free-ranging red-tailed hawks because of the vital role that the pectoral musculature plays in their ability to fly and thus the ability to be released after treatment and to survive.
Pharmacokinetic evaluation of antimicrobials requires performance of a validation assay specific to the targeted animal species. At the time of blood collection, subjects need to be free of medications that can potentially affect the validation process.6 Researchers can find it difficult to identify plasma samples obtained from captive red-tailed hawks that are free of other medications for assay validation. This difficulty has led to a need to find a suitable substitute for red-tailed hawk plasma to increase the ability to perform more pharmaceutical testing in this species in the future. Desirable traits for a substitute plasma donor include wide availability of the donor species, ability to collect a large volume of blood safely from each donor, and freedom from medication and disease. Chickens meet each of these qualifications, and chicken plasma is readily available for purchase from reliable laboratory vendors that maintain strict regulations regarding the health of their donors.
The purpose of the study reported here was to determine the dose of CCFA for red-tailed hawks that would exceed the MIC of raptor bacterial isolates, determine whether administration would adversely affect the pectoral musculature, and determine whether chicken plasma would be a suitable substitute for red-tailed hawk plasma in the validation process of the pharmacokinetic assay. We hypothesized that CCFA administered IM (pectoral region) once at 10 or 20 mg/kg would achieve plasma concentrations that exceeded the MIC for most raptor bacterial isolates for > 24 hours, that minimal to no adverse effects would be detected at the injection site, and that chicken plasma would be a suitable substitute for red-tailed hawk plasma in the assay validation process.
Materials and Methods
Animals
Seven permanently disabled but otherwise healthy red-tailed hawks housed at the University of California-Davis California Raptor Center were used for the study. All hawks were judged to be female on the basis of body weight (range, 1.1 to 1.6 kg). Health status was determined before the study began on the basis of results of physical examination, CBC, plasma biochemical analysis, and parasitologic examination of fecal specimens by use of a flotation technique. All hawks were > 1 year of age as determined by their plumage. The particular species was selected on the basis of their large size and their commonness as falconry birds and patients in wildlife rehabilitation centers. Hawks were housed in various sizes of flight pens, depending on their disability. They were fed thawed day-old chicks or rodents once daily and had free access to water. Food was withheld from hawks the night prior to study initiation. The experimental protocol was approved by the Institutional Animal Care and Use Committee at the University of California-Davis (No. 17281).
Drug administration and blood sample collection
In a crossover study design, hawks were randomly assigned by use of an online programa to first receive CCFAb at 10 mg/kg or 20 mg/kg, IM. A black permanent marker was used to draw a circle on the skin over the pectoral region at the site of injection prior to actual administration. The CCFA dose was injected into the pectoral muscle by use of a 3-mL Luer-lock syringe attached to a 25-gauge needle, with the needle inserted into the center of the black circle.
Hawks were manually restrained for venipuncture. Blood samples were collected from the right or left medial metatarsal, jugular, or basilic vein into heparinized tubes immediately prior to CCFA injection (0 hours) and 0.083, 0.16, 0.25, 0.5, 1, 2, 4, 8, 24, 48, 96, 120, 168, and 216 hours after CCFA injection. Collected samples were stored on ice until centrifugation for 10 minutes at 3,000 × g. Plasma was separated and stored in cryotubes at −80°C until analysis. After a 2-month washout period, CCFA administration and blood sample collection was repeated, with hawks receiving the other CCFA dose in the opposite pectoral muscle.
Each injection site was visually monitored once daily for bruising or other changes to the soft tissues and to ensure that the black circle remained easily visible until the final blood sample was collected for each administration session. On the day following the final blood sample collection (ie, day 10) of each administration session, hawks were prepared for collection of muscle biopsy specimens from the injection site. Food was withheld overnight, then hydromorphonec (0.3 mg/kg) was injected into the pectoral muscle opposite that for which the biopsy was planned. Anesthesia was induced by administration via face mask of 2% to 5% isoflurane in 100% oxygen at a flow rate of 1 L/min. Once anesthetic induction was achieved, an uncuffed, 3.0-mm endotracheal tube was placed into the trachea of each hawk. Anesthetic monitoring included measurements of end-tidal partial pressure of carbon dioxide, arterial blood pressure (via oscillometric device, with blood pressure cuff placed around the midfemoral region), oxygen saturation, and ECG variables.d Heart rate was monitored by means of a Doppler ultrasonographic flow detectore positioned over a basilic artery, and a heating pad was placed under each hawk to maintain body temperature during anesthesia. The feet of each hawk were wrapped to prevent self-injury.
The site identified for biopsy did not require plucking of the feathers and was aseptically prepared with dilute chlorhexidine solution.f A skin incision was created over the area of interest, and a 4-mm biopsy punchg was used to obtain specimens of muscle tissue at the injection site. For 1 hawk in which the first biopsy specimen was visibly white in color, a 2-mm biopsy punch was used instead to collect a specimen of muscle tissue deep to the biopsy site for the 4-mm punch. The muscle layer of each hawk was apposed with a single cruciate suture,h and the skin was apposed with 1 or 2 cruciate sutures.h Each hawk was allowed to recover from anesthesia while being manually restrained in an upright position, meloxicami (1 mg/kg) was administered PO, and crystalloid fluidj (50 mL/kg) was administered SC in the ventral or lateral inguinal region. The suture site was examined daily for 7 days, and meloxicam was administered twice per day for 7 days or until the sutures were removed, whichever came first.
Plasma sample analysis
Plasma samples were analyzed for concentrations of CFAEs, ceftiofur, and desfuroylceftiofur-related metabolites by means of high-performance liquid chromatography, as previously described.13 Dithioerythritolk (100 mg/0.5 mL of plasma) was added to plasma samples to cleave macromolecule-bound desfuroylceftiofur metabolites. Plasma samples were passed through a C18 solid-phase extraction cartridgel and derivatized with iodoacetamidem to yield desfuroylceftiofur acetamide. Additional cleanup was achieved by use of a strong cation exchange solid-phase extraction cartridge.n High-performance liquid chromatography was performed on the derivatized extracts isocratically (mobile phase solvent, 7% acetonitrile and 1% acetic acid with 90 mg of heptanesulfonic acid sodium salt/L; pH, 4.0) by use of a C18 column (particle size, 4-μm; column dimensions, 39 × 150 mm)o and UV (240 nm) detection.
Calibration standards for the assay were prepared by dilution of ceftiofur standardp in blank control chicken plasmaq purchased from a commercial vendor at concentrations from 0.2 to 20.0 μg/mL. Quality control samples of hawk plasma at 0, 0.4, 2.0, and 10.0 μg/mL were run with the study samples.
Interassay variability, as measured by relative SD, ranged from 4.1% to 4.8%, with a mean variability of 4.5%. Mean percentage recovery was 100.2%. Limit of detection was determined as 3 times the signal-to-noise ratio and limit of quantification as 10 times the signal-to-noise ratio at the expected retention time of derivatized desfuroylceftiofur acetamide in chromatograms of blank samples analyzed along with the QC sample, yielding values of 0.15 and 0.18 μg/mL, respectively.
Pharmacokinetic analysis
Pharmacokinetic analysis of time-versus-plasma concentration data was performed with commercial software.r Noncompartmental pharmacokinetic variables (t1/2λz Cmax, Tmax, AUC0–∞, extrapolated proportion of the AUC, and AUC0–∞/dose of CCFA) were determined for each dose for each hawk. Points at which plasma concentrations of CFAEs were higher than the MIC of ceftiofur were also determined.
MIC determination
Minimum inhibitory concentrations of ceftiofur were determined for bacteria that had been isolated from various clinical samples obtained from 55 raptors. The samples had been submitted to the William R. Pritchard Veterinary Medical Teaching Hospital Microbiology Laboratory at the University of California-Davis from January 2000 through June 2013. The MICs of ceftiofur determined for some (36/55) of the isolates had already been reported.6 For other isolates for which the MICs were unknown, bacteria were isolated on 5% sheep blood agar, MacConkey agar, or both and incubated at 35°C in 5% CO2. Identification of bacterial isolates was performed by use of conventional microbiological methods with spot tests, tubed media, and bacterial identification strips.s
The broth microdilution method was used to perform antimicrobial susceptibility testing in accordance with standards of the Clinical and Laboratory Standards Institute.14,t Briefly, 2 mL of brain-heart infusion broth was inoculated with 2 or 3 isolated bacterial colonies and incubated at 35°C without CO2 for 4 to 5 hours. The broth culture was then added dropwise to saline (0.9% NaCl) solution to achieve a McFarland standard of 0.5 as determined by use of a nephelometer.
Ten microliters of each suspension was diluted in 11 mL of cation-adjusted Mueller-Hinton broth that contained N-Tris(hydroxymethyl) methyl-2-aminoethane sulfonic acid; microwell platesl for determination of antimicrobial susceptibility were inoculated with 50 μL of diluted bacterial suspension/well. Plates were incubated at 35°C without CO2 overnight (16 hours). The MIC of ceftiofur for each bacterial isolate was determined.
For analysis of plasma CFAE concentrations achieved, consideration was made of the period that these concentrations were higher than a target MIC. Because some CFAEs are not active metabolites, the period that CFAEs were higher than the TPC was used to estimate the antibacterial activity from CCFA. Period above the target MIC was defined as the period that CFAE concentrations were higher than a target MIC. Period above the TPC was defined as the period during which the CFAE concentration was 4 times as high as the target MIC for ceftiofur; the multiple of 4 was chosen in an attempt to account for inadvertent inclusion in measurements of results for any inactive ceftiofur metabolites present, given that no information existed on inactive metabolites in any avian species. To establish the theoretic effectiveness of CCFA against various bacterial organisms, we determined the dose administration interval during which CFAE concentrations were maintained above TPC for 50% or 80% of the time for gram-positive and gram-negative bacteria, respectively.
Histologic evaluation of biopsy specimens
Biopsy specimens collected from the pectoral muscle of each hawk were bisected longitudinally. For histologic evaluation, half of each specimen was fixed in neutral-buffered 10% formalin, embedded in paraffin, and sectioned at 3 to 5 μm. Sections were placed on microscope slides and stained with H&E. The remaining half was embedded in water-soluble glycols and resins,u cooled to the freezing point in a 2-methylbutane bath in liquid nitrogen, and stored at −70°C. This step was taken to properly preserve tissue in the event that formalin-sensitive histochemical staining was necessary to characterize the lesion; however, such staining was not needed. Slides of skeletal muscle tissue were assessed via light microscope for the presence of inflammatory infiltrates, necrosis, fibroplasia or fibrosis, and dystrophic calcification. On the basis of the previously reported pattern of muscle damage that can occur after IM injections,15,16 severity of inflammatory changes was scored as follows: 0 = no inflammation, necrosis, or fibrosis; 1 = minimal to mild infiltration by inflammatory cells with some myofiber atrophy but no myofiber necrosis, fibroplasia, or fibrosis; 2 = moderate infiltration by inflammatory cells with scattered myofiber necrosis or interstitial fibroplasia or fibrosis; and 3 = moderate to severe infiltration by inflammatory cells with widespread myonecrosis or interstitial fibrosis, abscessation, or dystrophic mineralization.
Results
During both CCFA administration sessions (10 mg/kg or 20 mg/kg, IM), most red-tailed hawks remained healthy, with no clinically apparent adverse effects of drug administration; however, 1 hawk needed to be removed after the first session because of an iatrogenic capture injury that required antimicrobial treatment. All data from that hawk were excluded from the study, and the hawk was replaced with another healthy red-tailed hawk. The replacement hawk was used in both administration sessions to maintain 6 birds with full data sets in the study.
Standard curves representing CFAE concentrations in blank control plasma obtained from the red-tailed hawks prior to CCFA administration and in purchased plasma from chickens were linear (mean r2 for red-tailed hawk plasma, 0.9998; mean r2 for chicken plasma, 0.9995). Curves were identical as measured by the slope of the line, with mean intra-assay variability (relative SD) of 2.2% between the 2 plasma types, and mean interassay variability of 6.1%. To compare individual QC results, chromatographic peak areas from the standard curves and QC samples were combined. Mean intra-assay variability for that analysis was 5.0 (range, 2.1 to 15.3), and mean interassay variability was 15.9 (range, 6.5 to 30.8). Calculated concentrations were close to expected concentrations when comparing QC results to the curve made from data for the same type of plasma. Mean percentage difference between expected and actual values was −0.3% for red-tailed hawk plasma and −0.8% for chicken plasma, with mean variabilities of the calculated values of 1.7% and 2.0%, respectively. Calculated concentrations were not as close to expected values when compared with the curve from the other type of plasma (ie, hawk QC results with chicken standard curve or chicken QC results with hawk standard curve) but were acceptable. Mean percentage difference was still quite good at 1.2% for red-tailed hawk plasma and −2.1% for chicken plasma, but mean relative SDs of the calculated values were 8.9% and 9.6%, respectively.
Curves representing plasma concentration of CFAEs versus time were constructed for CCFA at 10 and 20 mg/kg doses (Figure 1). Noncompartmental pharmacokinetic data for both doses were summarized (Table 1).
Mean CFAE concentrations in plasma samples obtained from 6 red-tailed hawks (Buteo jamaicensis) at various points before (0 hours) and after IM administration of a single dose of CCFA at 10 mg/kg (A) or 20 mg/kg IM (B) in a randomized crossover study, with a 2-month washout period separating dose administration sessions and each hawk receiving both doses. The TPC of CFAE is represented by the dashed line at 4 μg/mL. Error bars represent the SD.
Citation: American Journal of Veterinary Research 76, 12; 10.2460/ajvr.76.12.1077
Mean ± SD values of noncompartmental pharmacokinetic variables for CFAEs in plasma samples obtained from 6 red-tailed hawks (Buteo jamaicensis) that received 1 dose of CCFA (10 mg/kg and 20 mg/kg, IM) in a crossover study design.
| Variable | 10 mg/kg | 20 mg/kg |
|---|---|---|
| AUC0–∞ (h·μg/mL) | 370 ± 88 | 958 ± 231 |
| Extrapolated percentage of AUC | 3.8 ± 1.4 | 4.8 ± 3.8 |
| t1/2λz (h) | 29 ± 9 | 50 ± 25 |
| Cmax (μg/mL) | 6.8 ± 2.3 | 15.1 ± 4.5 |
| Tmax (h) | 6.4 ± 2.2 | 6.7 ± 2.1 |
The MIC testing for ceftiofur revealed that 82% (45/55) of the clinical bacterial isolates from 55 raptors had an MIC ≤ 1.0 μg/mL. Only 1 additional isolate had a ceftiofur MIC between the cutpoints ≤ 1.0 μg/mL and ≤ 4.0 μg/mL, resulting in a total percentage of isolates with an MIC ≤ 4.0 μg/mL of 84%. Bacterial isolates with a ceftiofur MIC90 ≤ 1.0 μg/mL included coagulase negative Staphylococcus spp, Staphylococcus aureus, and Proteus mirabilis. Bacterial isolates with a ceftiofur MIC90 ≤ 4.0 μg/mL included Escherichia coli only. The MIC required to inhibit growth of 50% of bacterial isolates was ≤ 1.0 μg/mL for that bacterial species. The target MIC for ceftiofur was therefore determined as ≤ 1.0 μg/mL and the TPC as 4.0 μg/mL. For gram-negative organisms, plasma CFAE concentrations remained greater than the TPC for 36 and 96 hours for the 10 and 20 mg/kg doses, respectively, and for gram-positive organisms remained greater than the TPC for 45 and 120 hours, respectively.
Three of the 12 CCFA injection sites in the pectoral region of the hawks had evidence of minor bruising or crusting; crusting was identified at the site of needle insertion, and all crusting resolved within 3 days after injection. Before biopsies were performed 10 days after dose administration, drug injection sites of all hawks appeared grossly unremarkable, with no evidence of abscessation, swelling, crusting, or open lesions. All specimens from all but 1 hawk (after receiving the 20 mg/kg dose of CCFA) contained no grossly visible lesions. In the specimen from that hawk, a small (diameter, 1 to 3 mm), soft, pale yellow focus was visible. Histologic examination of the 12 specimens (1 for each hawk for each of the 2 CCFA doses) revealed no inflammation (2 specimens at the 10 mg/kg dose and 1 at the 20 mg/kg dose) or a small, concentrated focus of inflammation at the site of injection (4 specimens at the 10 mg/kg dose and 5 at the 20 mg/kg dose). No apparent necrosis, fibroplasia or fibrosis, or dystrophic mineralization was evident in any of the specimens.
Inflammation was characterized by a dense cluster of lymphocytes and histiocytes, with fewer plasma cells and granulocytes (Figure 2). Amounts of inflammatory cell infiltrate varied widely among specimens, comprising 0% to 50% of total specimen area. When present, inflammatory cells generally formed densely packed sheets of cells within the perimysium and often surrounded collagen bundles, small blood vessels, nerves, or variably sized (diameter, 10 to 200 μm) clear spaces that were interpreted as cottonseed oil droplets from the CCFA suspension. Smaller numbers of inflammatory cells extending into the endomysium between myofibers were common. Small aggregates of degenerate heterophils surrounded by epithelioid macrophages were identified in 3 of 12 biopsy specimens from both high and low dose groups (1 at the 10 mg/kg dose and 2 at the 20 mg/kg dose). Six of the 12 specimens (3/dose) contained multinucleated giant cells, which often bordered aforementioned clear spaces or degenerate heterophils. Despite clustering of inflammatory infiltrates directly around the site of injection as suggested by histopathologic findings, there was no evidence of myofiber degeneration or necrosis, fibroplasia or fibrosis, abscessation, or dystrophic mineralization in any specimen. Rarely, individual myofibers surrounded by areas of inflammation appeared to be slightly smaller in diameter, compared with diameters of neighboring myofibers that lacked inflammation (mild myofiber atrophy). In 3 of 12 specimens (2 at the 10 mg/kg dose and 1 at the 20 mg/kg dose), no inflammatory changes were identified. Tissue sections both with and without evidence of inflammation contained small amounts of finely granular mineral deposited in arterioles and small muscular arteries.
Photomicrographs of pectoral muscle tissue obtained from a red-tailed hawk 10 days after IM administration of CCFA at 10 mg/kg (A) or 20 mg/kg (B). A—Perimyseal inflammation is evident, with dense sheets of lymphocytes and histiocytes comprising most of the inflammatory cells. Regions of inflammation are surrounded by small, clear droplets (asterisks) and extend into the endomysium around individual myofibers. H&E stain; bar = 100 μm. B—Regions of inflammation surround small, clear droplets and atrophied myofibers (arrowheads). Small aggregates of degenerate heterophils (H) surrounded by epithelioid macrophages and multinucleated giant cells (arrows) were identified in biopsy specimens obtained from hawks after receiving both the 10 mg/kg (n = 1) and 20 mg/kg (3) doses. H&E stain; bar = 50 μm.
Citation: American Journal of Veterinary Research 76, 12; 10.2460/ajvr.76.12.1077
Discussion
Ceftiofur crystalline-free acid administered IM once at a dose of 20 mg/kg had a longer duration of antimicrobial action in red-tailed hawks in the present study than has been reported for CCFA administered IM to birds of other species, with a mean t1/2λz of CCFA of 50 hours. On the other hand, mean t1/2λz of CCFA for the 10 mg/kg dose was similar among red-tailed hawks in the present study (29 hours) and American black ducks (32 hours)6 and helmeted guineafowl (29 hours)11 in other studies. The greater t1/2λz for the 20 mg/kg dose in red-tail hawks versus other bird species was most likely attributable to the extent and rate of absorption of the larger dose. This finding should be considered when multiple doses of CCFA are administered IM to red-tailed hawks.
Mean Tmax of CFAE for the red-tailed hawks in the present study (6.4 hours for 10 mg/kg, IM and 6.6 hours for 20 mg/kg, IM) was much shorter than that reported for American black ducks (24 hours for 10 mg/kg, IM)6 and helmeted guineafowl (19.3 hours for 10 mg/kg, IM).11 Mean Cmax for the 10 mg/kg dose in red-tailed hawks was similar to that reported for the same dose in helmeted guineafowl, but mean Cmax for the 20 mg/kg dose in red-tailed hawks was more similar to that reported for the 10 mg/kg dose in American black ducks.
To compensate for detection of inactive ceftiofur metabolites with high-performance liquid chromatography and because the ratio of circulating ceftiofur to its less active metabolites in birds is unknown, the TPC in the present study (4 μg/mL) was established as 4 times as high as the target MIC for ceftiofur (1 μg/mL). Some studies17,18 conducted to determine pharmacokinetics of ceftiofur in cattle and swine have involved a target MIC of 0.2 μg/mL, despite the fact that the reported MIC of ceftiofur is 0.03 μg/mL for many bacterial species commonly recovered from these animals. Results of a study19 in which antimicrobial activities of ceftiofur and its most active metabolite, desfuroylceftiofur, were investigated indicate that both have similar activities against gram-negative bacteria but that ceftiofur is 2 to 8 times as active as is desfuroylceftiofur against certain gram-positive bacteria. Given the high proportion of bacterial isolates from raptors for which the ceftiofur MIC90 was < 1.0 μg/mL in the present study, a TPC of 4 μg/mL might be expected to be active against many of the bacterial isolates that were susceptible to ceftiofur. These findings suggested that CCFA could be administered IM to red-tailed hawks every 36 and 45 hours at 10 mg/kg and every 96 and 120 hours at 20 mg/kg to treat infections with gram-negative and gram-positive organisms, respectively. Even at the 10 mg/kg dose, this administration interval could considerably decrease the number of physical restraint episodes required to complete a course of antimicrobial treatment. However, because of the lack of data regarding active and inactive metabolites in avian species and the existing data for mammals,19 a higher TPC of CFAE might be necessary for red-tailed hawks. Additional studies are needed to identify ratios of active and inactive metabolites in the particular avian species in which CCFA is to be administered to support accurate dosage recommendations.
To achieve the best therapeutic outcome in animals of a certain species, pharmacokinetic and efficacy data are needed on antimicrobial administration in that species. When species-specific pharmacokinetic data are lacking, allometric scaling of existing pharmacokinetic data for other species might be another option for determining therapeutic doses. Ceftiofur crystalline-free acid is rapidly metabolized; therefore, whether it would be an appropriate candidate for allometric scaling is unknown given that many drugs with extensive metabolism are not candidates for that method.20 Pharmacokinetics of approximately 75% of drugs is reportedly not scalable across multiple species.21 Indeed diet, anatomic features, and metabolism can differ extensively across various avian species, which further complicates possibilities for allometric scaling of drug pharmacokinetics. Additional studies are needed to critically evaluate whether allometric scaling of CCFA pharmacokinetic variables can be used to apply results for one avian species to other avian species.
Injection of CCFA into pectoral muscle at either dose in the present study resulted in a mild, localized, inflammatory response that was not grossly visible in most hawks. Despite the histologic finding that some biopsy specimens contained a substantial number of inflammatory cells, the overall intensity of inflammation within the pectoral muscle specimens was interpreted as mild for 2 reasons. First, inflammation was not associated with other lesions such as myodegeneration or fibrosis; only mild myofiber atrophy was occasionally observed. Second, an inflammatory infiltrate was completely lacking in 3 of 12 specimens, presumably because the inflammation was so minor and localized to the injection site that it might have been missed during specimen collection and processing. Detection of multinucleated giant cells within several specimens was consistent with a foreign-body reaction. Because multinucleated cells were commonly identified surrounding oil droplets, this reaction was likely in response to the cottonseed oil used in the CCFA suspension.
Overall, the intensity and nature of the inflammatory reaction observed was considered an appropriate immunologic response to injection of an exogenous substance and did not result in overt secondary complications. The cause and importance of early mineralization in the tunica muscularis of some arteries and arterioles were unknown. These lesions were identified within both inflamed and histologically normal muscle tissue and, as such, may have represented a degenerative change unrelated to CCFA administration.
Little to no inflammation at the sites of injection 10 days after CCFA administration suggested that, after IM administration of a single dose, little to no residual pathological effects might be expected within the pectoral musculature. However, it is unknown whether the flight of birds released prior to 10 days after administration might be compromised or whether administration of multiple doses would result in more substantial lesions. The mild inflammation that was observed may have resulted from the use of a 25-gauge needle, which was smaller than the 20- to 22-gauge needle that is typically used to administer the thick ceftiofur formulation. Additional studies are necessary to understand whether multiple IM injections of CCFA increase the potential for pathological effects in muscles used for flight.
Acquiring an adequate supply of control plasma from exotic animal species for routine pharmacokinetic analysis can be challenging. Such plasma is not commercially available, and the physical size and number of available donor animals are commonly limited. To overcome these obstacles, we investigated whether plasma samples obtained from healthy chickens could be used as a substitute for plasma samples obtained from untreated red-tailed hawks for control samples in pharmacokinetic studies. Three separate extractions were performed with each type of plasma, each consisting of a standard curve and set of QC samples. When chicken plasma was used as a matrix for the control and standard curve samples, results were similar to when red-tailed hawk plasma was used.
Limitations of the study reported here included the small sample size (6 red-tailed hawks). The smallest number of hawks possible was used to complete the pharmacokinetic analysis, and a larger number would have been advantageous for evaluation of variability among individual hawks. However, the SDs of the data acquired were small, suggesting the effect of that variability on the results would have been small. Additionally, because of the small volume (diameter, 4 mm) of muscle tissue obtained during the biopsy procedure and the inherent difficulty collecting specimens from an injection site 10 days after CCFA injection, biopsy specimens may have included skeletal muscle tissue at some distance from the exact point of drug injection. As a result, some biopsies may have captured only a portion or none of the inflammation incited by the CCFA injection, accounting for the observed differences in amounts of inflammatory infiltrates. We attempted to use a consistent biopsy protocol to increase confidence that specimen collection included the injection site. Although the techniques used may have made it difficult to differentiate subtle differences in the severity of inflammation between the low and high doses of CCFA administered, they nonetheless provided good evidence that neither dose incited extensive inflammation, given that tissue immediately adjacent to the injection site was histologically normal.
Additional research should be conducted to identify ratios of active and inactive metabolites of ceftiofur in specific avian species to help identify the most appropriate TPC for each species and to evaluate the pharmacokinetics of CCFA after administration of multiple doses. Repeated IM administration of this drug may cause adverse effects to the musculature that were not identified in the present study, and this supposition should also be evaluated. Effects of repeated IM administration would be particularly important for the 20 mg/kg dose of CCFA because the t1/2λz for that dose appeared to be influenced more by the extent and rate of absorption than the 10 mg/kg dose. Furthermore, because of differences among avian species in pharmacokinetic responses, it would be beneficial to evaluate the pharmacokinetics of CCFA in other raptorial species prone to distress during handling that could benefit from a decrease in drug administration and, hence, handling frequency.
Acknowledgments
Supported by the Morris Animal Foundation (grant No. D14ZO-808) and the Center for Companion Animal Health, University of California-Davis.
The authors thank Bret Stedman and volunteers at the California Raptor Center for the care of the birds used in this study and Scott Wetzlich for assistance with plasma CFAE concentration analysis.
ABBREVIATIONS
| AUC0–∞ | Area under the plasma concentration versus time curve from 0 hours to infinity |
| CCFA | Ceftiofur crystalline-free acid |
| CFAE | Ceftiofur free-acid equivalent |
| Cmax | Maximum plasma concentration |
| MIC | Minimum inhibitory concentration |
| MIC90 | Minimum inhibitory concentration required to inhibit growth of 90% of bacterial isolates |
| QC | Quality control |
| t1/2λz | Terminal-phase half-life |
| Tmax | Time to maximum plasma concentration |
| TPC | Therapeutic plasma concentration |
Footnotes
Research Randomizer, version 4.0, Geoffrey C. Urbaniak and Scott Plous, Middletown, Conn. Available at: www.randomizer.org/form.htm. Accessed Dec 8, 2012.
Excede swine injectable (100 mg/mL), Zoetis, Madison, NJ.
Fort Dodge Animal Health, Overland Park, Kan.
Digicare LifeWindow Lite, Boynton Beach, Fla.
Doppler flow detector model 811-BL, Parks Medical Electronics Inc, Aloha, Hawaii.
Nolvasan solution, Fort Dodge Animal Health, Fort Dodge, Iowa.
Integra Miltex standard biopsy punch, Integra LifeSciences, Plainsboro, NJ.
Polydioxanone-II, 4–0 taper needle, Ethicon, Somerville, NJ.
Metacam, Boehringer Ingelheim, St Joseph, Mo.
Lactated Ringer solution, Hospira, Lake Forest, Ill.
Dithioerythritol, Acros Organics, Morris Plains, NJ.
C18 SPE cartridge, Thermo Scientific, Bellefonte, Pa.
Iodoacetamide, Acros Organics, Morris Plaines, NJ.
SCX SPE cartridge, Agilent Technologies, Folsom, Calif.
Nova-Pak C18 Column, Waters Corp, Milford, Mass.
Ceftiofur Standard, Vetranal, Riedel-de Haën, Seelze, Germany.
Chicken plasma, Lampire Biological Laboratories, Pipersville, Pa.
Phoenix WinNonlin, version 6.3, Pharsight, Sunnyvale, Calif.
API BioMerieux, Durham, NC.
Sensititre, ThermoFisher, Waltham, Mass.
Tissue-Tek OCT compound, Sakura Finetek USA Inc, Torrance, Calif.
References
1. Flammer K. Antibiotic drug selection in companion birds. J Exot Pet Med 2006; 15: 166–176.
2. Bird JE, Miller KW, Larson AA, et al., Pharmacokinetics of gentamicin in birds of prey. Am J Vet Res 1983; 44: 1245–1247.
3. Bloomfield RB, Brooks D, Vulliet R., The pharmacokinetics of a single intramuscular dose of amikacin in red-tailed hawks (Buteo jamaicensis). J Zoo Wildl Med 1997; 28: 55–61.
4. Harrenstein L, Tell L, Vulliet R, et al., Disposition of enrofloxacin in red-tailed hawks (Buteo jamaicensis) and great horned owls (Bubo virginianus) after a single oral, intramuscular, or intravenous dose. J Avian Med Surg 2000; 14: 228–236.
5. Robbins PK, Tell LA, Needham ML, et al., Pharmacokinetics of piperacillin after intramuscular injection in red-tailed hawks (Buteo jamaicensis) and great horned owls (Bubo virginianus). J Zoo Wildl Med 2000; 31: 47–51.
6. 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: 620–627.
7. Tell L, Harrenstien L, Wetzlich S. Pharmacokinetics of ceftiofur sodium in exotic and domestic avian species. J Vet Pharmacol Ther 1998; 21: 85–91.
8. Freedom of information summary. Original new animal drug application. EXCEDE for swine (ceftiofur crystalline free acid) sterile suspension. NADA 141–235. New York: Pharmacia & Upjohn Co, 2006.
9. FDA. Freedom of Information summary. Supplemental new animal drug application. EXCEDE sterile suspension. Ceftiofur crystalline free acid. Horses. NADA 141–209. Washington, DC: FDA, 2009.
10. FDA. Freedom of Information summary. Supplemental new animal drug application. EXCEDE for Swine. Ceftiofur crystalline free acid. Sterile suspension. Swine. NADA 141–235. Washington, DC: FDA, 2010.
11. 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: 1514–1518.
12. Flammer K., Use of enrofloxacin (baytril) in psittacine birds; in Proceedings. 3rd Int Vet Symp Fluoroquinolones 1999;107–109.
13. Jaglan PS, Cox BL, Arnold TS, et al., Liquid chromatographic determination of desfuroylceftiofur metabolite of ceftiofur as residue in cattle plasma. J Assoc Off Anal Chem 1990; 73: 26–30.
14. Clinical and Laboratory Standards Institute., Performance standards for antimicrobial disk and dilution susceptibility tests for bacteria isolated from animals: approved standard. VET01–A4. Wayne, Pa: Clinical and Laboratory Standards Institute, 2013.
15. Rasmussen F. Tissue damage at the injection site after intramuscular injection of drugs. Vet Sci Commun 1978; 2: 173–182.
16. Boray JC. Local damage caused by the intramuscular injection of carbon tetrachloride in sheep and cattle. Aust Vet J 1965; 4: 291–294.
17. FDA. Freedom of Information summary. Naxcel sterile powder. Ceftiofur sodium. NADA 140.338. Washington, DC: FDA, 2004.
18. Brown SA, Chester ST, Speedy AK, et al., Comparison of plasma pharmacokinetics and bioequivalence of ceftiofur sodium in cattle after a single intramuscular or subcutaneous injection. J Vet Pharmacol Ther 2000; 23: 273–280.
19. 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: 332–336.
20. Hunter RP, Isaza R. Concepts and issues with interspecies scaling in zoological pharmacology. J Zoo Wildl Med 2008; 39: 517–526.
21. Riviere JE, Martin-Jimenez T, Sundloff SF, et al., Interspecies allometric analysis of the comparative pharmacokinetics of 44 drugs across veterinary and laboratory animal species. J Vet Pharmacol Ther 1997; 20: 453–463.
