The veterinary literature is replete with studies into the efficacy of both IVRLP and IORLP in horses for treatment of injury or disease in the carpal, tibiotarsal, metacarpophalangeal, metatarsophalangeal, proximal interphalangeal, and distal interphalangeal joints.1–11 The use of various vessels, antimicrobials, and indwelling catheters has also been investigated.2–7,10,12–20 Aside from application in human and equine patients, successful use of RLP has been described in a wallaby (Wallabia bicolor),21 lesser kudu (Tragelaphus imberbis),21 and Asian elephant (Elephas maximus)22 as well as rabbits23 and cattle.24 Among avian species, RLP has been described for only 3 brown pelicans (Pelecanus occidentalis),25 a common loon (Gavia immer),25 and a rooster.26 All 5 of those patients received systemic treatment in addition to 1 or 2 IVRLPs.25,26 To our knowledge, RLP (neither IV nor IO) has not been validated for use in avian species.
In birds, bacterial infections of bones, joints, and the surrounding soft tissues can cause challenges for successful treatment. Severe bacterial infections often impair blood perfusion of and antimicrobial penetration into affected tissues,23 which can delay or prevent healing. Failure of antimicrobials to penetrate infected tissues increases the risks for osteomyelitis and septicemia. Orthopedic injuries are common in companion, wild, and zoological-collection birds subsequent to falls, window strikes, predator or prey bites, gunshot wounds, and other penetrating foreign materials. Most penetrating wounds and traumatic fractures are open and contaminated. Any soft tissue infection in the distal aspect of a limb (distal limb) of a bird has the potential to spread into the bone because there is often limited soft tissue covering the bones. Therefore, swift and effective treatment of open fractures and penetrating wounds is necessary to prevent progression of the injury to osteomyelitis. In birds with primary osteomyelitis or septic arthritis, aggressive antimicrobial treatment is indicated to prevent further loss of bone or exacerbation of septicemia, which could necessitate amputation or euthanasia. Amputation might be an acceptable alternative for some pet birds, but amputation in a wild bird often leads to euthanasia because the patient cannot be released back into the wild. Thus, there is a need for the development of more effective and useful alternatives to successfully treat birds with open and contaminated wounds or traumatic fractures.
Regional limb perfusion is a well-established method for delivering antimicrobials at concentrations greater than the minimum inhibitory concentration for many common bacterial pathogens into the synovium and osseous and soft tissue structures of the distal limbs of horses.1,6,8,9,11,12,14–17 The purpose of the study reported here was to assess whether IVRLP and IORLP could be used to deliver clinically relevant concentrations of ceftiofur sodium into the synovial fluid of the tibiotarsal-tarsometatarsal joint of chickens (ie, an avian model) and to determine whether one of those RLP methods was superior to the other.
Materials and Methods
Animals
All study procedures were reviewed and approved by the Tufts University Institutional Animal Care and Use Committee. Twelve adult hens retired from laying eggs (retired layers) were obtained for the study. The birds weighed between 1.6 and 2.6 kg and were considered healthy on the basis of results of a physical examination. Each physical examination included documentation of the subject's body weight, heart rate, and respiratory rate; quantification and evaluation of urofeces production; food consumption; and any eggs laid. Birds were individually housed in large metal cages (66 × 81 × 76 cm) lined with pine shavings in an indoor climate-controlled university facility. They were provided water and a pelleted ration designed for layers ad libitum and mealworms intermittently as enrichment. The birds were maintained on a 12-hour light and 12-hour dark cycle. Birds were allowed to acclimate to their surroundings for 1 week prior to study initiation, and a physical examination was performed on each bird daily.
Study design
Each bird was assigned to receive ceftiofur sodiuma (2 mg/kg) by the IVRLP (n = 4), IORLP (4), or IM (control; 4) route once daily for 6 consecutive days. The day when the first dose of ceftiofur was administered was designated day 1. All birds were anesthetized for ceftiofur administration and blood and synovial fluid sample collection. Each bird underwent a physical examination as previously described prior to each anesthesia event. Birds were individually weighed to ensure that the appropriate dose of ceftiofur was injected. They were also examined for signs of inflammation or infection at the injection site or sites. Birds with evidence of lameness; swelling, hematoma, or cellulitis that precluded identification of the medial metatarsal vein; or substantial erythema, bleeding, discharge, or malodor at any injection site were removed from the study and administered appropriate treatment. Birds with mild local inflammation or a hematoma that did not preclude identification and venipuncture of the medial metatarsal vein were retained in the study. After study completion, all birds were adopted by an animal sanctuary where they were allowed to live out their natural lives. No meat or eggs from the chickens were used for human consumption during or after the study.
Anesthesia
Food but not water was withheld from each bird for 4 hours before each anesthesia event. Anesthesia was induced with isoflurane in 95% to 98% oxygen delivered via a face mask until the bird was sedate enough for orotracheal intubation. Each bird was intubated with a 3.0-, 3.5-, or 4.0-mm uncuffed endotracheal tube, and anesthesia was maintained with isoflurane in oxygen. The bird was positioned in dorsal recumbency and manually ventilated at approximately 10 breaths/min. While the bird was anesthetized, heart rate was monitored by use of a Doppler ultrasonographic probe, which was positioned over the brachial artery. Isoflurane administration was discontinued after treatment and sample collection were complete for the day, and the bird was allowed to recover from anesthesia. Oxygen continued to be delivered until the swallow reflex was regained, at which time it was discontinued, and the endotracheal tube was removed.
Ceftiofur administration
Each bird was anesthetized and received ceftiofur (2 mg/kg) by the assigned route once daily for 6 consecutive days. For birds assigned to the IVRLP and IORLP groups, all 6 doses of ceftiofur were administered in the same leg. Within a given group, the left leg was used in 2 birds, and the right leg was used in 2 birds. This was chosen by alternating between the left and right legs. Birds in the control group were administered the drug in the pectoral muscles, alternating sides for each successive dose.
For birds in the IVRLP group, a photograph of the medial metatarsal vein of the leg selected for ceftiofur injection was obtained before administration of the first dose to serve as a referent to aid assessing whether repetition of the procedure resulted in any visually evident changes in the vessel and surrounding tissues. For each IVRLP procedure, a Penrose drain was used as a tourniquet and was positioned around the selected leg at the midtibiotarsal level and secured with a hemostat. The skin overlying the medial metatarsal vein was disinfected with chlorhexidine scrub and alcohol. A 25-gauge butterfly catheter was aseptically placed in the vein with proper positioning confirmed by the presence of a steady flash of blood in the catheter. The calculated dose of ceftiofur was diluted with sufficient sterile saline (0.9% NaCl) solution to achieve an injection volume of 3 mL, which was then injected through the catheter followed by 0.3 mL (the volume of the butterfly catheter) of sterile saline solution to ensure that the entire dose was administered. The catheter was removed, and a bandage was placed over the injection site. The tourniquet was left in place for 10 minutes after injection of the ceftiofur to allow the drug to perfuse into the tissues distal to it. Then, the tourniquet was removed followed by removal of the bandage over the injection site. This order of events was important because removal of the bandage prior to removal of the tourniquet could result in hematoma formation, which could impair access to the vessel for subsequent injections.
For birds in the IORLP group, feathers were plucked from the proximal aspect of the tibiotarsal region, and the underlying skin was aseptically prepared with chlorhexidine scrub and alcohol. A 2% lidocaine solution (0.05 to 0.1 mL) was injected into the periosteum of the proximal portion of the tibiotarsal bone with a 25-gauge needle attached to a 1-mL syringe to provide local analgesia. A Penrose drain was used as a tourniquet and was positioned around the selected leg at the midfemoral level and secured with a hemostat. A sterile 22-gauge, 1.5-inch spinal needle was aseptically placed into the proximal aspect of the tibiotarsal bone to serve as an indwelling IO catheter. A catheter cap was attached to the needle hub, and the catheter was secured with a drop of tissue glue at the catheter-skin interface and tape tabs that were sutured to the skin. The calculated dose of ceftiofur was diluted with sufficient sterile saline solution to achieve an injection volume of 3 mL, which was injected through the catheter followed by 0.3 mL of heparinized saline solution to ensure that the entire dose was administered and to keep the catheter patent. The tourniquet was left in place for 10 minutes to allow the drug to perfuse into the tissues distal to it. Then, the tourniquet was removed, and the leg was lightly wrapped with a bandage to cover, secure, and protect the indwelling IO catheter, which remained in place for the duration of the treatment period (6 days).
Sample collection
Synovial fluid samples were collected from a tibiotarsal-tarsometatarsal joint of each bird 15 minutes after ceftiofur administration on days 1 and 6. For birds in the IVRLP and IORLP groups, synovial fluid samples were collected from the tibiotarsal-tarsometatarsal joint of the leg used for ceftiofur administration. For each bird in the control group, the tibiotarsal-tarsometatarsal joint used for sample collection was selected in the same manner as that used to select which leg would be used for ceftiofur administration to birds in the IVRLP and IORLP groups. At each designated sampling time, a synovial fluid sample (0.05 to 0.3 mL) was aseptically collected from the lateral aspect of the tibiotarsal-tarsometatarsal joint with a 22-gauge needle attached to a 1-mL syringe as described.27 The sample was placed in a cryovial and stored frozen at −80°C until analysis.
From all birds, a blood sample (approx 2 to 3 mL) was collected by venipuncture of the right jugular vein 15 minutes after ceftiofur administration on days 1, 3, and 6. The blood samples were placed in sterile blood collection tubes containing heparin as an anticoagulant. Blood samples were centrifuged, and the plasma was harvested. Each plasma sample was divided into two 1-mL aliquots, which were placed in separate cryovials and stored frozen at −80°C until analysis.
For each bird, 1 aliquot of plasma from each sample acquisition time was submitted to the University of Miami Avian and Wildlife Laboratory for biochemical analysis. All synovial fluid samples and the remaining plasma samples were submitted to the University of Tennessee Pharmacology Laboratory for determination of ceftiofur concentration.
Quantification of plasma and synovial fluid ceftiofur concentration
Plasma and synovial fluid ceftiofur concentrations were determined by use of reversed-phase HPLC. The HPLC system consisted of a separation moduleb and UV detector.c The mobile phase consisted of 0.1% trifluroacetic acid in water (A) and 0.1% trifluroacetic acid in acetonitrile (B). The mobile phase gradient started at 90% A and 10% B, adjusted to 75% A and 25% B over 25 minutes, and then transitioned back to the initial conditions over 3 minutes. Separation was obtained on a C18 columnd (length, 250 mm; internal diameter, 4.6 mm; particle size, 5 μm) with a guard column,e both of which were maintained at ambient temperature. The flow rate was 1.0 mL/min, and the UV detector was set at 265 nm.
Ceftiofur was extracted from plasma and synovial fluid samples by use of a slightly modified derivatization method that converts ceftiofur and all desfuroylceftiofur metabolites to desfuroylceftifur acetamide.28 Briefly, frozen synovial fluid and plasma samples were thawed and vortexed. Then, for each sample, 100 μL was transferred to a clean test tube to which 15 μL of internal standard (cefotaxime, 100 μg/mL) and 7 mL of 0.4% dithioerythritol in borate buffer were added. The tube was placed in a warm water bath (50°C) for 15 minutes. The tube was removed from the water bath and allowed to cool to room temperature (approx 22°C), after which 1.5 mL of iodoacetamide buffer was added. The solution was passed through an extraction column.f The column was eluted with a 5% glacial acetic acid-methanol solution, which was evaporated to dryness under a steady stream of nitrogen gas. The sample was reconstituted in 200 μL of mobile phase, and 50 μL of the resulting mixture was injected into the HPLC system.
Standard curves were prepared by spiking untreated plasma samples with ceftiofur to create standards with linear concentrations of the drug that ranged from 0.1 to 100 μg/mL. The spiked standards were processed in the same manner as the plasma samples. The mean percentage of ceftiofur recovery was 99%. Intra-assay variability ranged from 0.7% to 4.5%, and interassay variability ranged from 3.6% to 8.8%. The lower limit of quantification for ceftiofur was 100 ng/mL.
Statistical analysis
For each treatment group (IVRLP, IORLP, and control), descriptive statistics were generated for plasma biochemical variables and synovial fluid and plasma ceftiofur concentrations. For this study, a ceftiofur concentration > 1 μg/mL was considered the therapeutic threshold for both plasma and synovial fluid samples; this assumption was made on the basis of results of an in vivo study29 in which ceftiofur sodium was administered to mice. For each biochemical variable and the synovial fluid ceftiofur concentration, the difference between the values on days 6 and 1 was calculated for each bird. That difference was then used as the outcome variable in a general linear model and compared among the 3 treatment groups. A mixed linear model was used to compare plasma ceftiofur concentration over time and among the 3 treatment groups. The model included fixed effects for treatment group and day (1, 3, or 6) and a random effect for bird to account for repeated measures within subjects. For all models, the Tukey adjustment was used when post hoc pairwise comparisons were necessary. All analyses were performed with statistical software,g and values of P < 0.05 were considered significant.
Results
Birds
For the IVRLP group, bruising around the medial metatarsal vein was evident in 2 birds on day 3 and all 4 birds on day 6. However, the bruising was only moderate in the most severely affected birds and did not prevent the treatment regimen from being completed in any of the birds. No adverse effects associated with ceftiofur administration were observed in any of the birds in the IORLP and control groups.
Effect of treatment on plasma biochemical variables
Descriptive statistics for select plasma biochemical variables for the birds in the 3 treatment groups on days 1 and 6 were summarized (Table 1). The plasma total protein concentration was mildly increased from the upper limit of the reference range30 for individual birds in all 3 treatment groups on both sampling days, but the mean total protein concentration was within the reference range for all 3 treatment groups on both days 1 and 6. The mean plasma CK activity was increased from the upper limit of the reference range for the IVRLP and IORLP groups on day 1 and for all 3 groups on day 6; however, the mean difference in CK activity between days 6 and 1 did not differ significantly among the 3 groups. The mean plasma calcium concentration increased from day 1 to day 6 for the control and IVRLP groups and decreased for the IORLP group. The magnitude of the mean difference in plasma calcium concentration between days 6 and 1 was significantly less for the IVRLP group (P = 0.016) and greater for the IORLP group (P < 0.001), compared with that for the control group. The mean AST activity increased between days 1 and 6 for all 3 treatment groups. The magnitude of the mean difference in AST activity between days 6 and 1 for the IORLP was significantly (P = 0.01) greater than that for the control group but did not differ significantly between the IVRLP and control groups.
Descriptive statistics for select plasma biochemical variables on days 1 and 6 for 12 healthy adult hens (layers) that received ceftiofur sodium (2.2 mg/kg) by the IVRLP (n = 4), IORLP (4), or IM (control; 4) route once daily for 6 consecutive days.
| IVRLP | IORLP | Control | ||||||
|---|---|---|---|---|---|---|---|---|
| Variable | Reference range30 | Day | Mean ± SD | Range | Mean ± SD | Range | Mean ± SD | Range |
| Glucose (mg/dL) | 217–300 | 1 | 222.50 ± 12.40 | 206.0–235.0 | 241.25 ± 13.82 | 227.0–260.0 | 236.25 ± 18.30 | 219.0–262.0 |
| 6 | 227.50 ± 6.14 | 220.0–235.0 | 235.50 ± 15.33 | 219.0–256.0 | 228.25 ± 25.49 | 206.0–264.0 | ||
| Calcium (mg/dL) | 13.2–23.7 | 1 | 17.85 ± 5.91 | 11.0–24.3 | 21.38 ± 3.12 | 19.4–26.0 | 23.20 ± 6.31 | 15.0–30.2 |
| 6 | 18.18 ± 6.16 | 12.2–24.3 | 12.90 ± 2.28 | 11.2–16.2 | 26.75 ± 4.03 | 23.4–32.1 | ||
| Total protein (g/dL) | 3.3–5.5 | 1 | 4.35 ± 0.62 | 3.8–5.2 | 5.35 ± 1.81 | 3.8–7.8 | 5.90 ± 1.41 | 4.6–7.4 |
| 6 | 4.20 ± 0.75 | 3.2–5.0 | 4.75 ± 0.44 | 4.2–5.2 | 6.05 ± 1.11 | 5.0–7.2 | ||
| Uric acid (mg/dL) | 2.5–8.1 | 1 | 3.35 ± 0.87 | 2.2–4.2 | 2.53 ± 0.85 | 1.5–3.4 | 2.75 ± 1.16 | 1.8–4.4 |
| 6 | 4.95 ± 1.07 | 3.6–5.9 | 3.53 ± 1.03 | 2.4–4.5 | 3.00 ± 1.58 | 1.5–4.6 | ||
| AST (U/L) | 45–422 | 1 | 178.50 ± 26.91 | 160.0–218.0 | 157.50 ± 7.68 | 151.0–166.0 | 164.25 ± 20.73 | 146.0–194.0 |
| 6 | 209.25 ± 45.70 | 178.0–277.0 | 286.25 ± 63.85 | 209.0–365.0 | 191.75 ± 26.59 | 158.0-–23.0 | ||
| CK (U/L) | 110–580 | 1 | 1,862.50 ± 1,975.50 | 664.0–1,811.0 | 640.75 ± 184.18 | 492.0–893.0 | 406.75 ± 86.13 | 316.0–510.0 |
| 6 | 861.00 ± 224.55 | 654.0–1,160.0 | 1,749.75 ± 1,096.07 | 827.0–3,256.0 | 930.25 ± 280.19 | 687.0–1,274.0 | ||
| Phosphorus (mg/dL) | 2.8–7.9 | 1 | 3.68 ± 0.74 | 2.7–4.5 | 3.68 ± 1.11 | 2.4–4.7 | 4.30 ± 1.27 | 2.7–5.7 |
| 6 | 3.75 ± 0.47 | 3.1–4.2 | 2.90 ± 0.78 | 1.9–3.8 | 4.55 ± 1.28 | 3.4–5.8 | ||
The day when the first dose of ceftiofur was administered was designated day 1.
Synovial fluid and plasma ceftiofur concentrations
Descriptive statistics for plasma (Table 2) and synovial fluid (Table 3) ceftiofur concentrations were summarized. Mean plasma ceftiofur concentration was significantly associated with treatment group (P < 0.001) but not day. The mean plasma ceftiofur concentration for the IORLP group was significantly greater than that for the IVRLP (P = 0.014) and control (P = 0.002) groups, but the mean plasma ceftiofur concentration did not differ significantly (P = 0.40) between the IVRLP and control groups.
Descriptive statistics for plasma ceftiofur concentration (μg/mL) on days 1, 3, and 6 for the birds of Table 1.
| Day | Statistic | IVRLP | IORLP | Control |
|---|---|---|---|---|
| 1 | Mean ± SD | 6.00 ± 3.00 | 16.40 ± 10.58 | 1.75 ± 0.79 |
| Median (range) | 6.35 (2.20–9.10) | 12.55 (8.50–32.00) | 1.85 (0.80–2.50) | |
| 3 | Mean ± SD | 6.75 ± 3.54 | 14.55 ± 4.14 | 4.95 ± 0.70 |
| Median (range) | 7.70 (1.70–9.90) | 16.25 (8.40–17.30) | 4.85 (4.20–5.90) | |
| 6 | Mean ± SD | 9.03 ± 3.09 | 13.55 ± 2.72 | 6.83 ± 1.09 |
| Median (range) | 8.45 (5.90–13.30) | 13.55 (10.50–16.60) | 6.50 (5.90–8.40) |
Plasma ceftiofur concentrations > 1 μg/mL were considered therapeutic.
See Table 1 for remainder of key.
Descriptive statistics for synovial fluid ceftiofur concentration (μg/mL) on days 1 and 6 for the birds of Table 1.
| Day | Statistic | IVRLP | IORLP | Control |
|---|---|---|---|---|
| 1 | Mean ± SD | 206.93 ± 229.12 | 2.58 ± 1.19 | 1.30 ± 0.85 |
| Median (range) | 104.50 (46.–469.40) | 2.90 (0.90–3.60) | 1.30 (0.30–2.30) | |
| 6 | Mean ± SD | 118.10 ± 117.27 | 6.83 ± 5.32 | 1.53 ± 0.28 |
| Median (range) | 112.25 (16.50–231.40) | 5.45 (2.30–14.10) | 1.55 (1.20–1.80) |
Synovial fluid ceftiofur concentrations > 1 μg/mL were considered therapeutic.
See Table 1 for remainder of key.
The mean synovial fluid ceftiofur concentration increased between days 1 and 6 for the control and IORLP groups but decreased for the IVRLP group (Table 3). The magnitude of the mean difference in synovial fluid ceftiofur concentration between days 6 and 1 for the IVRLP group was significantly (P < 0.001) greater than that for both the IORLP and control groups but did not differ significantly (P = 0.59) between the IORLP and control groups.
Discussion
Results of the present study indicated that IVRLP resulted in a significantly greater concentration of ceftiofur in the synovial fluid of the ipsilateral tibiotarsal-tarsometatarsal joint of chickens than either IORLP or IM administration of the same dosage of the drug. Additionally, following IVRLP, the synovial fluid ceftiofur concentration was several times that in plasma. Although serial administration of ceftiofur by IVRLP resulted in mild to moderate bruising of the medial metatarsal vein, that bruising did not prevent completion of the prescribed treatment regimen, and no other adverse effects were observed in the birds assigned to that treatment group. These findings suggested that IVRLP might be a safe and effective technique for administration of antimicrobials to birds with joint infections, contaminated wounds, pododermatitis, and other musculoskeletal infections of the distal limb.
In equine practice, RLP techniques are commonly used to treat horses with distal-limb infections as well as for prophylaxis following surgical repair of wounds to the distal limbs.3–6,10,19,31 Local phlebitis of the vein used for injection is the most common adverse effect observed in horses following IVRLP32 and was also observed for the birds of the present study. However, in the present study, phlebitis was fairly mild and did not prevent the procedure from being repeated as prescribed.
Intravenous RLP and IORLP techniques are used to achieve clinically relevant concentrations of various drugs to local tissues of horses.10,11,33 Aside from horses, RLP has been successfully used in an elephant,22 wallaby,21 and lesser kudu21 as well as cattle,24 rabbits,23 humans,34 and various avian species.25,26 Additionally, one of the investigators (SEK) of the present study has frequently used RLP for at least 10 years to successfully treat distal-limb infections in birds of many species. Given that the anatomy of the distal limb varies among bird species, validation of both the IVRLP and IORLP techniques in birds provides avian practitioners with alternatives so that RLP can be performed regardless of distal-limb anatomy. Aside from anatomic concerns, the primary limiting factor for IVRLP is vessel damage from successive venipunctures; therefore, avian practitioners may choose to use the less invasive IVRLP technique for patients that require short-term treatment regimens and the more invasive IORLP technique for patients that require long-term treatment regimens.
In the present study, the 15-minute interval between ceftiofur administration and collection of synovial fluid and blood samples was chosen on the basis of data obtained following ceftiofur administration to horses,35 chickens, and Amazon parrots.36,37 Ceftiofur concentrations in radiocarpal synovial fluid remain > 1 μg/mL (the therapeutic threshold of ceftiofur for many bacterial pathogens in many species) for > 24 hours after IVRLP of 2 g of ceftiofur to healthy adult horses.35 For the horses of that study,35 the synovial fluid ceftiofur concentration peaked at 30 minutes after IVRLP but exceeded 1 μg/mL by 5 minutes after IVRLP. Also, the synovial fluid ceftiofur concentration was significantly greater than the plasma ceftiofur concentration within 30 minutes after administration of the drug by IVRLP, whereas the plasma ceftiofur concentration was significantly greater than the synovial fluid ceftiofur concentration within 30 minutes after administration of the drug by the IV (systemic) route.35 Intravenous administration of ceftiofur failed to achieve therapeutic drug concentrations in synovial fluid for > 8 hours. Plasma ceftiofur concentrations remained > 1 μg/mL for > 4 hours after SC and IM injection of the drug to chicken poults and adult psittacines (cockatiels and Amazon parrots), respectively.36 In both the chicken poults and adult psittacines of that study,36 the plasma ceftiofur concentration peaked at 30 minutes and was > 1 μg/mL as soon as 3 minutes after drug administration. Although allometric scaling and extrapolation of drug pharmacokinetics among species have substantial limitations, those techniques are often the only recourse avian and exotic animal practitioners have to help guide the development of treatment regimens until the scientific literature provides more accurate and objective data. Given that the metabolic rate of birds is generally greater than that of horses and on the basis of the previously outlined findings,35,36 it seemed reasonable to assume that 15 minutes would be sufficient time for therapeutic concentrations of ceftiofur to be achieved in the synovial fluid following IVRLP and IORLP administration of the drug for the chickens of the present study.
Many birds of the present study were actively laying eggs, although the number of eggs laid on a daily basis decreased over the 6-day treatment period. We believe that the significant differences in plasma calcium concentration observed among the 3 treatment groups were likely caused by fluctuation of systemic calcium concentrations associated with the egg-laying patterns of individual birds rather than an effect of the ceftiofur administration route.38 The mean plasma AST activity increased between days 1 and 6 for all 3 treatment groups, which was most likely attributable to tissue trauma associated with handling of the birds for ceftiofur administration. However, the mean difference in AST activity between days 6 and 1 for the IORLP group was significantly greater than that for the control group but did not differ significantly between the IVRLP and control groups. The increase in AST activity was greatest for the IORLP group, which was likely a reflection of the inherent minor muscle and bone trauma associated with placement of an IO catheter. The mean plasma CK activity was near or exceeded the upper limit of the reference range for all 3 treatment groups on both days 1 and 6 but did not differ among the 3 groups. This was an expected finding because CK is a reactive enzyme that increases rapidly following handling and injections.
In horses, the antimicrobial concentration achieved in plasma is greater than that in synovial fluid following drug administration by IORLP, whereas the antimicrobial concentration achieved in synovial fluid is greater than that in plasma following drug administration by IVRLP.33,39 The disparity in antimicrobial distribution between the IORLP and IVRLP techniques is probably associated with pressure differences between the 2 administration routes.33,39 During IORLP, the drug is injected into the medullary cavity of a bone, which is a noncollapsible and fairly low-pressure environment even distal to a tourniquet. This allows passive absorption of the drug into the systemic circulation. In contrast, during IVRLP, the drug is injected directly into a regional vein, which is readily collapsible and is typically a high-pressure environment owing to tourniquet placement proximal to the injection site. That high pressure facilitates diffusion of the antimicrobial into the surrounding tissues, which in turn decreases the amount of drug retained within the systemic circulation.
In the present study, the mean plasma ceftiofur concentration for the IORLP group was consistently greatest and was significantly greater than that for the IVRLP and control groups at all 3 sample acquisition times. The same type of tourniquet was used for both the IORLP and IVRLP procedures, but it was placed at the midfemoral level during the IORLP and the midtibiotarsal level during the IVRLP. It is possible that placement of the tourniquet at the midfemoral level was ineffective in restricting venous outflow from the leg. In birds, the muscle density and anatomic adduction of the femur make it a particularly challenging bone to access for tourniquet placement. Thus, the absorption dynamics of ceftiofur from the bone marrow cavity following IORLP may have been similar to those following systemic IV injection and contributed to the high plasma ceftiofur concentrations observed for that group relative to those for the IVRLP and control groups. It is also possible that, for the birds in the IORLP group, the bandage applied to the tibiotarsal region to protect the indwelling IO catheter might have caused sufficient soft tissue compression to decrease local venous availability of ceftiofur and impaired diffusion of the drug along the pressure gradient into the synovial fluid. That possibility was supported by the fact that the mean synovial fluid ceftiofur concentration for the IORLP group was significantly lower than that for the IVRLP group. Unlike horses, the optimal tourniquet for use with RLP in birds has not been determined. The application of more robust compression to the leg by the tourniquet during and after IORLP may have resulted in synovial fluid ceftiofur concentrations closer to those achieved for the IVRLP group. However, even in horses with optimal tourniquet placement, the synovial fluid antimicrobial concentration achieved after IORLP is generally less than that achieved after IVRLP.33,39
The bactericidal efficacy of ceftiofur is associated with the duration that the drug concentration in the target tissue remains above the minimum inhibitory concentration for the causative pathogen.29,35 For all 3 treatment groups of the present study, the mean ceftiofur concentration in both plasma and synovial fluid exceeded the therapeutic threshold (1 μg/mL) of ceftiofur for many bacterial pathogens at all sample acquisition times. The synovial fluid ceftiofur concentration varied substantially among birds, especially those in the IVRLP and IORLP groups. Nevertheless, the median synovial fluid ceftiofur concentration for the IVRLP group ranged from approximately 21 to 72 times that for the IORLP and control groups, which suggested that IVRLP may be the preferred route of antimicrobial administration when penetration of the drug into local tissues is desired. The mean synovial fluid ceftiofur concentration for the IORLP group was numerically, but not significantly, greater than that for the control group at all sample acquisition times. Failure to detect a significant difference between the mean synovial fluid ceftiofur concentrations for the IORLP and control groups might have been caused by a lack of power owing to the small sample size (n = 4 birds/group); a significant difference may have been detected if the study population had been larger.
It is important to note that the present study was a pilot project, the intent of which was to validate the use of IVRLP and IORLP in an avian research model to provide information for future studies involving companion and wild bird species only. We chose to use ceftiofur sodium because it is a commonly used antimicrobial in companion and zoological avian medicine, is fairly nonirritating to tissues in the event of extravasation during IV or IO administration, and has limited adverse effects in species with a renal-portal system.36 Chickens were selected as the model species because they are readily available and there are pharmacokinetic data for ceftiofur in chickens as well as for turkeys, ducks, and several psittacine species.36,37,40 In the United States, ceftiofur sodium is labeled for the control of early death associated with susceptible Escherichia coli isolates in day-old chicks and turkey poults.41 Other formulations of ceftiofur (eg, ceftiofur hydrochloride and ceftiofur crystalline-free acid) are not approved for use in poultry. The FDA prohibits extralabel use of ceftiofur sodium in major food-producing species, including chickens and turkeys; however, ceftiofur sodium use in companion and wild birds is permitted under AMDUCA. In short, ceftiofur was administered to the chickens of the present study in an extralabel manner that was not in compliance with AMDUCA. The Food Animal Residue Avoidance and Depletion Program does not provide withdrawal intervals for animals that are administered drugs in a manner that is expressly prohibited by the FDA or is not in compliance with AMDUCA and generally recommends that such animals or products (eg, eggs or milk) from those animals never be used for human consumption. The chickens used for the present study were adopted by an animal sanctuary and never entered the human food chain.
Results of the present study indicated that ceftiofur concentrations believed to be therapeutic against most bacterial pathogens could be achieved in synovial fluid samples obtained from the tibiotarsal-tarsometatarsal joint of chickens following IVRLP and IORLP of ceftiofur (2.2 mg/kg) once daily for 6 consecutive days. These findings suggested that IVRLP and IORLP may be safe and effective techniques for administering antimicrobials to birds with joint infections, contaminated wounds, pododermatitis, and other musculoskeletal infections of the distal limb. Further research is necessary to determine the optimal volume of perfusate, tourniquet material, and tourniquet-applied pressure for IVRLP and IORLP in various species of birds commonly maintained as companion animals or in zoological collections. Given the limitations of allometric scaling between species, additional studies are warranted to determine the pharmacokinetics of commonly used antimicrobials following RLP in wild birds and birds commonly maintained as companion animals and in zoological collections to help guide development of appropriate antimicrobial treatment regimens for birds with distal-limb infections.
Acknowledgments
The authors thank Sherry Cox, PhD, Pharmacology Laboratory, College of Veterinary Medicine, University of Tennessee, and Rachel Turner, DVM, and Cheryl Stout, V'19, Cummings School of Veterinary Medicine, Tufts University, for technical assistance.
ABBREVIATIONS
| AST | Aspartate aminotransferase |
| CK | Creatine kinase |
| HPLC | High-performance liquid chromatography |
| IO | Intraosseous |
| IORLP | Intraosseous regional limb perfusion |
| IVRLP | IV regional limb perfusion |
| RLP | Regional limb perfusion |
Footnotes
Naxcel, Zoetis Inc, Parsippany, NJ.
2695 Separation Module, Waters Corp, Milford, Mass.
2487 UV Detector, Waters Corp, Milford, Mass.
Symmetry C18 Column, Waters Corp, Milford, Mass.
Symmetry Guard Column, Waters Corp, Milford, Mass.
Oasis HLB Extraction Column, Waters Corp, Milford, Mass.
SAS, version 9.2, SAS Institute Inc, Cary, NC.
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