Plasma concentrations of enrofloxacin after single-dose oral administration in loggerhead sea turtles (Caretta caretta)

Journal of Zoo and Wildlife Medicine 36(4): 628–634, 2005 Copyright 2005 by American Association of Zoo Veterinarians

PLASMA CONCENTRATIONS OF ENROFLOXACIN AFTER SINGLE-DOSE ORAL ADMINISTRATION IN LOGGERHEAD SEA TURTLES (CARETTA CARETTA) Elliott Jacobson, D.V.M., Ph.D., Dipl. A.C.Z.M., Ronald Gronwall, D.V.M., Ph.D., Lara Maxwell, D.V.M., Ph.D., Dipl. A.C.V.C.P, Kelly Merrit, B.S., and Glenn Harman, B.S.

Abstract: Plasma concentrations and pharmacokinetics of enrofloxacin were determined in 12 loggerhead sea turtles (Caretta caretta) after oral administration. Six turtles in group 1 and group 2 received enrofloxacin at 10 mg/kg and 20 mg/kg of body weight, respectively. Blood was collected from the cervical sinus before administration and at timed intervals up to 168 hr following administration. Plasma concentrations of enrofloxacin were determined using a microbiologic assay. The mean peak plasma concentration (Cmax) was 4.07 mg/ml and 21.30 mg/ml for groups 1 and 2, respectively. Plasma levels were detectable at 168 hr postadministration, with mean values of 0.380 mg/ml for group 1 and 2.769 mg/ml for group 2. The mean elimination half-life for enrofloxacin was 37.80 hr for group 1 and 54.42 hr for group 2. These findings indicated that enrofloxacin is absorbed following oral administration in loggerhead sea turtles, and blood levels are maintained up to 168 hr following administration. Key words: Loggerhead sea turtle, Caretta caretta, enrofloxacin, oral, plasma concentrations, pharmacokinetics.

INTRODUCTION Following their arrival in clinical medicine in the later 1980s, the fluoroquinolones have become a widely used group of synthetic antimicrobials in veterinary medicine.28 The fluoroquinolone enrofloxacin has become a commonly used antimicrobial in reptile medicine because of its activity against both gram-positive and gram-negative aerobic bacteria and few adverse effects associated with its use.12 The efficacy of enrofloxacin is enhanced in several species by the formation of an active metabolite, ciprofloxacin, which exhibits a similar potency and spectrum of activity.28 Although initial studies to determine scientifically derived dosages of enrofloxacin in reptiles focused on the injectable formulation,22 it subsequently became clear that local irritation and soft tissue necrosis can occur following enrofloxacin injections.22,30 This resulted in studies designed to assess uptake following oral administration. The pharmacokinetics of enrofloxacin disposition following oral administration have been investigated in green iguanas (Iguana iguana),20 savannah monitors (Varanus exanthematicus),11 and red-eared sliders (Trachemys scripta elegans).14 In these species, therapeutic plasma concentrations of enrofloxacin were achieved, but From the College of Veterinary Medicine, University of Florida, Gainesville, Florida 32610, USA (Jacobson, Gronwall, Merrit); College of Veterinary Medicine, Oklahoma State University, Stillwater, Oklahoma 74078, USA (Maxwell); and Clearwater Marine Aquarium, 249 Windward Passage, Clearwater, Florida 33767, USA (Harman). Correspondence should be addressed to Dr. Jacobson.

disposition varied markedly among species, indicating that extrapolation between reptile species is likely to result in inaccurate dosing of enrofloxacin. Although wild loggerhead sea turtles (Caretta caretta) frequently require antimicrobial therapy when brought into oceanariums and rehabilitation facilities, few antimicrobial dosing studies have been performed. The disposition of parenterally administered ceftazadime and florfenicol have been investigated in loggerhead sea turtles.26,27 Because long courses of antimicrobial therapy are frequently required in reptile medicine, oral administration of therapeutics in food is a practical, well-tolerated method of drug administration.13 Unfortunately, there is only a single oral anti-microbial drug-dosing study in sea turtles.19 Because of enrofloxacin’s use in other species of reptiles, and uptake following oral administration in certain turtles,14 we decided to perform an oral dosing study in loggerhead sea turtles. The pharmacokinetics of enrofloxacin following a single high or low orally administered dose in loggerhead sea turtles was determined. MATERIALS AND METHODS Turtles Twelve healthy loggerhead sea turtles (five female, two male, and five of undetermined sex) were used in this study. Turtles were maintained in indoor pools at Clearwater Marine Aquarium, Clearwater, Florida. Water temperature was 25–298C. The diet consisted of locally purchased squid (Opalesgus loligo). This project was approved by the University of Florida Institutional Animal Care and Use Committee. All samples were collected under

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the authorization of Florida Fish and Wildlife Conservation Commission Marine Turtle Permit No. 086. Experimental design Before initiation of the study, appropriate enrofloxacin dosages and blood sampling intervals were determined from a small pilot study. Two turtles (undetermined sex; 40 kg and 50 kg in body weight) received 10 mg/kg or 20 mg/kg of orally administered enrofloxacin in food, as described below. Following the analysis of samples collected during the pilot study, sea turtles were divided into two dosage groups. Group 1 consisted of six loggerhead sea turtles that weighed 40.6–90.9 kg (four female and two of unknown sex), and group 2 consisted of six loggerhead sea turtles that weighed 28.6–130.9 kg (one female, two male, and three of unknown sex). Turtles in groups 1 and 2 received single doses of enrofloxacin at 10 mg/kg and 20 mg/kg of body weight, respectively. Enrofloxacin tablets (136 mg; Bayer Corporation, Shawnee Mission, Kansas 66201, USA) were combined with a meal of squid at a dosage rate that was appropriate for each turtle. After removal of the tentacles and viscera from a squid, the required number of enrofloxacin tablets were inserted into the body cavity. To enclose the drug, the ends of the squid were overlapped and sutured with 3–0 absorbable suture material (PDS, Ethicon, Sommerville, New Jersey 08876, USA). The enrofloxacin-containing squid was offered to the designated turtle and was readily accepted. Blood samples (5 ml) were collected into tubes containing lithium heparin (Fisher Scientific Inc., Pittsburgh, Pennsylvania 15275, USA) from the dorsal cervical sinus of each turtle just before administration of enrofloxacin and at 1, 3, 6, 9, 12, 24, 48, 72, 96, 120, 144, and 168 hr after dosing. All tubes were initially placed on crushed ice and were centrifuged within 15 min of collection. Plasma was transferred to cryotubes and frozen in a container of liquid nitrogen. Samples were transported to the University of Florida, where they were stored at 2808C until drug analysis. Enrofloxacin assays Plasma samples collected during the pilot study were analyzed for enrofloxacin and ciprofloxacin by high-performance liquid chromatography (HPLC) with fluorescence detection.7 In contrast, antimicrobial activity of samples collected from the main portion of the study were determined by an agar gel well diffusion microbiological assay1 with Klebsiella pneumonia (ATCC 10031, American Type Culture Collection, Manassas, Virginia

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20108, USA) as the assay organism. One milliliter of the bacterial suspension was grown in Mueller Hinton Broth (Fisher Scientific Inc., Pittsburgh, Pennsylvania 15275, USA) and adjusted to an optical density of 0.5 at 550 nm. The suspension was added to tempered Mueller Hinton agar (Fisher Scientific Inc., Pittsburgh, Pennsylvania 15275, USA) and distributed evenly into the assay plates. The plates were allowed to solidify for 45 min, and 0.5mm wells were punched. On the day that the experimental assays were performed, samples for a calibration curve were prepared by adding pure enrofloxacin (Bayer Corporation, Shawnee Mission, Kansas 66201, USA) to unmedicated loggerhead sea turtle plasma. Wells were filled with 50 ml of nine enrofloxacin standards ranging in concentrations from 8 to 0.03 mg/ml; no blank was used. The standard curve was calculated using a logarithmiclinear model. Experimental samples were processed similarly to those for the calibration curve. The agar plates were incubated for 16 hr at 378C. Zones of bacterial inhibition were measured to the nearest 0.1 cm. Each sample or standard was assayed in triplicate, and mean values for three measurements of the zone diameters were determined. The lower limit of quantification (LLOQ) of the assay was 0.03 mg/ml of enrofloxacin and the coefficient of variation for the assay ranged from 4.2% for the 8 mg/ml standard to 10% for the 0.03 mg/ml standard. None of the postdosing plasma samples was below the LLOQ. The microbiological assay measures enrofloxacin and any active metabolites such as ciprofloxacin. Because the calibration curve was derived using pure enrofloxacin, assay results represent enrofloxacin equivalents (in mg/ml). In reporting and discussing the results of this study, enrofloxacin and enrofloxacin equivalents are used interchangeably. Pharmacokinetic and statistical analysis Pharmacokinetic analysis: Pharmacokinetic analysis was performed using a noncompartmental approach.8 Plasma enrofloxacin concentrations for each trial were used to fit the following equation using a computer program that minimized the weighted sums of squared deviations3: CP 5 C1e2l1t 1 C2e2l2t 2 (C1 1 C2)e2l3t The model solution was used to determine the initial decay rate (l1), the terminal decay rate (l2), the apparent absorption rate (l3), area under the curve (AUC0–` 5 C1/l1 1 C2/l2 2 [C1 1 C2]/l3), and area under the moment curve (AUMC 5 C1/l12 1 C2/ l22 2 [C1 1 C2]/l32). From these values, the halflife (t½ 5 ln[2]/l) and mean residence time (MRT

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Table 1. Mean (6 standard deviation) concentrations of plasma enrofloxacin equivalents (mg/ml) for two groups of six loggerhead sea turtles receiving oral enrofloxacin at either 10 mg/kg or 20 mg/kg of body weight. Group 1 (10 mg/kg) Time (hr)

Mean

SD

0.00 1.00 3.00 6.00 9.00 12.00 24.00 48.00 72.00 96.00 120.00 144.00 168.00

0 0.108 0.695 1.636 2.166 2.538 3.746 2.322 1.497 1.052 0.715 0.474 0.380

0 0.210 0.954 1.656 2.062 2.063 0.750 0.801 0.617 0.489 0.409 0.276 0.244

Group 2 (20 mg/kg) Mean

0 2.845 4.576 6.505 10.516 16.634 21.054 15.162 9.457 6.703 4.688 3.832 2.769

SD

0 4.134 5.464 5.482 6.664 8.182 6.375 6.543 3.025 0.876 0.514 0.411 0.351

5 AUMC/AUC) were determined. The AUC over the first 24 hr after enrofloxacin administration, AUC0–24, was calculated using the above model. Because the amount of drug absorbed was not known, only the volume of distribution combined with bioavailability (F) was determined according to the relationship: Vdarea/F 5 dose/AUC/l2. To test for dosage proportionality, the dose-normalized AUC obtained after a single oral dosage of 10 mg/kg of enrofloxacin was compared to that obtained following a dosage of 20 mg/kg, using a onetailed, two-sample t-test (SigmaStat 3.0.1; SPSS Inc., Chicago, Illinois, USA). In order to examine the source of any observed departure from dose proportionality, effect of dosage group on body mass, Vd,area/F, the apparent elimination half-life of enrofloxacin, and the enrofloxacin disposition rate constants l1, l2, and l3, were also tested using twotailed, two-sample t-tests. In order to meet requirements for normality, the disposition rate constants were natural log transformed. The equivalence of the variances between groups and the normality of all tested variables were verified using the Levene Median and Kolmogorov-Smirnov tests, respectively. For all tests, differences were considered significant at P 5 0.05.

Figure 1. Plasma enrofloxacin equivalents (mg/ml) versus time (hr) in loggerhead sea turtles following administration of a single oral 10 mg/kg or 20 mg/kg dose. Error bars are standard deviations (SD) of the mean of measured plasma enrofloxacin concentrations.

Plasma concentrations of enrofloxacin for group 1 (10 mg/kg) and group 2 (20 mg/kg) are presented in Table 1 and Figure 1, and the pharmacokinetic parameters are in Table 2. The mean observed Cmax was 4.1 mg/ml and 21.3 mg/ml and the Tmax was 20.0 and 26.0 hr for groups 1 and 2, respectively. Plasma concentrations were maintained above the LLOQ up to the last sampling time of 168 hr. The elimination half-lives for groups 1 and 2 were 37.8 hr and 54.4 hr, respectively. From a computer algorithm,3 mean values for AUC0–` and AUC0–24 Table 2. Pharmacokinetic parameters (mean 6 SD) after oral administration of 10 mg/kg and 20 mg/kg of enrofloxacin to loggerhead sea turtles. Parametera

Tmax (hr) Cmax (mg/ml) T½(l1) (hr) T½(l2) (hr) T½(l3) (hr) AUC0–` (mg·hr/ml) AUC0–24 (mg·hr/ml) MRT (hr) Vdarea/F (ml/kg)

10 mg/kg

20.0 4.1 4.5 37.8 6.1 261 77 68 2167

6 6 6 6 6 6 6 6 6

6.2 0.8 3.3 12.6 2.0 104 36 15 477

20 mg/kg

26.0 21.3 6.1 54.4 7.0 1799 481 89 933

6 6 6 6 6 6 6 6 6

11.8 6.6 5.2 23.3 4.6 115 276 33 739

RESULTS All loggerhead sea turtles in this study were fed squid before, throughout, and after the study period. All had a good appetite, and no detrimental effects were seen following administration of either dose of enrofloxacin. All turtles were released following the end of the study.

a Tmax, time of observed maximal plasma concentration; Cmax, maximal observed plasma concentration; T½(l1), half-life of initial drug decay; T½(l2), half-life of terminal drug decay; T½(l3), half-life of the apparent drug absorption; AUC0–`, total area under the plasma concentration-versus-time curve from 0 to infinity; AUC0–24, total area under the plasma concentration-versus-time curve from 0 to 24 hr after administration of the drug; MRT, mean residence time; Vdarea/F, volume of distribution area/bioavailability.

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were calculated to be 260.9 mg•hr/ml and 77.3 mg•hr/ml, respectively for group 1 and 1799.0 mg•hr/ml and 481.4 mg•hr/ml, respectively, for group 2. The dose-normalized AUC obtained after a dosage of 20 mg/kg was higher than that associated with a dosage of 10 mg/kg (P , 0.05). Additionally, Vd,area/F was lower in turtles that had received the higher dosage of enrofloxacin (P , 0.05). However, body mass, the apparent elimination half-life of enrofloxacin, and the enrofloxacin disposition rate constants l1, l2, and l3 did not differ significantly between the two dosage groups (P . 0.05). DISCUSSION Although enrofloxacin has several clinically advantageous attributes, such as a broad spectrum of activity and a wide therapeutic index, that have resulted in its common use in reptiles with certain bacterial infections, the injectable form can be extremely irritating, and this always needs to be considered when this route of administration is selected. One of us (ERJ) has seen focal to diffuse areas of necrosis at intramuscular injection sites in snakes and chelonians. This has been reported by others.22,30 Furthermore, one of us (ERJ) has observed excessive salivation in juvenile Galapagos tortoises (Geochelone elephantopus) that were administered a single intramuscular (i.m.) injection. This reaction also has been reported in a large Galapagos tortoise that received enrofloxacin intramuscularly.4 Because loggerhead sea turtles are the most common marine turtle brought into rehabilitation facilities in Florida, and necrosis following intramuscular injection has been seen by one of us in sea turtles given i.m. injections of enrofloxacin (ERJ), we decided to evaluate two doses of oral enrofloxacin in loggerhead sea turtles. The low dose (10 mg/kg) was selected from studies with enrofloxacin in other turtles.14 The higher dose was arbitrarily selected. In this study, enrofloxacin was measured in plasma of loggerhead sea turtles using a microbiological assay. Although this assay is not capable of differentiating between enrofloxacin and any active metabolites, such as ciprofloxacin, microbiological assays do allow quantification of the antimicrobial contributions from both the parent drug and any active drug metabolites. Although HPLC assays can be used to determine the concentration of both enrofloxacin and ciprofloxacin in plasma, this assay was not locally available to us following the conclusion of the pilot study. The microbiologic assay may overestimate the actual amount of enrofloxacin in the sample, but in studies using both HPLC and a microbiological assay for determining enrofloxa-

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cin in serum of horses, it was found that most of the antimicrobial activity resided in the parent drug, and the half-life of the antibacterial activity paralleled the half-lives of enrofloxacin and ciprofloxacin.15 In addition, a relatively small portion of enrofloxacin is metabolized to ciprofloxacin in mammals5,9 and in those few reptiles studied.10,12,14,20,30 Similar results were obtained when pharmacokinetic studies of enrofloxacin disposition were performed in dogs using microbiological and HPLC methods.16 Finally, the data collected during the pilot study indicated that ciprofloxacin plasma concentrations were less than 15% of the value of enrofloxacin concentrations at all time points at which samples were collected. This relatively low percentage suggests that enrofloxacin contributes most of the antimicrobial activity resulting from the administration of enrofloxacin to loggerhead sea turtles. Thus, total antimicrobial activity, as measured by the microbiological assay, was considered adequate in this study to determine an appropriate dose of enrofloxacin to orally administer to loggerhead sea turtles. Advantageous pharmacokinetic properties of fluoroquinolones in mammals include rapid absorption following oral administration, a large volume of distribution, penetrating nearly every tissue and cell in the body, and an extended elimination halflife.28 Although peak concentration in monogastric mammals is reached in around 2 hr, the mean Tmax in the loggerhead sea turtles was 20 hr for the 10 mg/kg group and 26 hr for the 20 mg/kg group. The turtles received enrofloxacin tablets in squid, and possibly this affected the uptake. Food may delay the occurrence of peak concentrations28 or inhibit absorption.2 Prolonged uptake has been reported in other reptiles following oral administration. In savanna monitors administered enrofloxacin orally, the Tmax was 36 hr,11 and in American alligators it was 55 hr.10 This is in contrast to studies in red-eared sliders and green iguanas, where Tmax following oral administration was 5 hr and 2.7 hr, respectively.14,20 Prolonged absorption was also noted in a previous study of loggerhead sea turtles in which praziquantel was also administered orally in squid13, and a biphasic uptake was seen in Ridley sea turtles administered itraconazole in either squid or capelin.19 Slow absorption rates following oral administration of drugs to loggerhead sea turtles may occur commonly in this species or may be a result of squid administration. Additionally, to prevent the enrofloxacin tablets from being extruded when the turtles were fed, the ends of the squid had to be sutured. This probably further delayed the uptake.

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Each turtle received the same dose of enrofloxacin per kilogram of body weight, and the turtles comprising groups 1 and 2 did not differ significantly in mean body weights. However, turtles in both groups varied considerably in weight (41–91 kg for the group 1 and 29–131 kg for group 2). The variation in plasma levels at each sampling point may partially be explained by metabolic differences between turtles that differ in weight. The apparent elimination half-life for the loggerhead sea turtles was 37.80 and 54.42 hr, respectively, for the 10 and 20 mg/kg dosing groups. This is considerably longer than the half-life for mammals but within the range for other reptiles.10,11,14,20,22,23,25,30 The half-life in red-eared sliders that received oral enrofloxacin at 10 mg/kg was 33 hr and was comparable to mean values of loggerhead sea turtles (37.80 hr) that received the same dose. An advantage of such long half-lives is that the frequency of administration of enrofloxacin in reptiles is reduced as compared to mammals. Plasma concentration findings revealed that loggerhead sea turtles that received 20 mg/kg body weight had approximately five times the mean maximum blood levels of enrofloxacin compared to the group that received 10 mg/kg. Likewise, the mean AUC determined from enrofloxacin dosing at 20 mg/kg was approximately six times that obtained from the 10 mg/kg dosing schedule. There are several possible reasons for this departure from doseproportionality. First, because this was not a crossover study, and different turtles (of similar weight range) were used in the two groups, the nonproportionality may have been attributed to interindividual turtle differences. Because turtles had to be released following the first dosing study, a crossover study could not be performed to test this possibility. Alternatively, the rates of enrofloxacin absorption, enrofloxacin elimination, or binding of enrofloxacin to peripheral tissues may be capacitylimited processes at the dosage range studied. For example, in dogs receiving subcutaneously administered enrofloxacin at dosage rates of 1.25, 2.5, and 25 mg/kg, the apparent elimination half-life increased from approximately 2 hr at the lower dosage rates to 6.4 hr at 25 mg/kg.21 Similarly, the elimination of ofloxacin in pigs was shown to be a capacity-limited process at dosage rates of 3, 10, and 30 mg/kg.24 Although the mean apparent elimination half-life of the loggerhead sea turtles in the 20 mg/kg enrofloxacin dosage group was longer than that of the 10 mg/kg group, this parameter did not differ significantly (P . 0.05) between the two dosage groups. Similarly, the disposition rate constants, l1, l2, and l3, determined for each dosing

group also did not differ (P . 0.05), indicating that the saturation of absorption, distribution, or elimination mechanisms could not explain the lack of dose-proportionality. Although Vdarea/F of enrofloxacin was lower in the turtles that had received the higher dose rate, this was not surprising, as Vdarea/F 5 dose/AUC/l2. Unfortunately, the current study was not designed to investigate the source of nonlinear kinetics following oral dosages of enrofloxacin to loggerhead sea turtles. As an intravenous dose of enrofloxacin was not administered, the disposition rate constants associated with the oral administration of enrofloxacin could not be unambiguously assigned to absorption and elimination rate constants, complicating the testing of these rate constants for capacity-limited processes. Additionally, a feeding effect associated with the placement of tablets in squid may also have enhanced the bioavailability of enrofloxacin in the turtles receiving a higher dosage of enrofloxacin. Therefore, the source of the lack of dose-proportionality in the present study is currently unclear and requires further study. A crossover study involving a range of dosages of intravenously and orally administered enrofloxacin, with determination of plasma enrofloxacin and ciprofloxacin concentrations by HPLC, would serve to answer the questions raised by the present study about dose-proportionality of orally administered enrofloxacin in loggerhead sea turtles. Historically, the minimum inhibitory concentration (MIC) of a drug has been used to recommend antimicrobial dosing regimens. When targeting Pseudomonas aeruginosa, peak plasma concentrations that are four times the MIC exert both a suprainhibitory and a postantibiotic effect.18 Other studies have determined that bactericidal concentrations for fluoroquinolones are reached at 8–10 times the MIC.6 More recently, the following three pharmacodynamic parameters (pharmacokinetic/ bacteriologic hybrids) have been advocated as the best way to accurately predict therapeutic outcome: (1) the time that the simulated drug concentrations remain above the minimum inhibitory concentration (MIC) of the susceptible organism (t . MIC); (2) the maximum drug concentration to MIC ratio (Cmax/MIC); and (3) the area under the simulated serum time curve to MIC ratio (AUC/MIC).17,29 Because quinolone antibiotics are generally considered to have concentration-dependent bactericidal activity, maximum drug concentration/MIC and AUC/MIC ratios have been identified as possible pharmacodynamic predictors of clinical and microbiologic outcome as well as bacterial resistance.29 Data from in vitro models indicated that high Cmax/MIC values are important in preventing emer-

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gence of resistance, whereas AUC/MIC is the best predictor of antibacterial effect.17 It has been suggested that Cmax/MIC ratios . 10 or AUC/MIC ratios of 100–125 are required for quinolones to have clinical and microbiologic success and to limit the development of bacterial resistance. Although MIC values of bacteria isolated from loggerhead sea turtles are not available, MIC data for other chelonians has been reported. In a study with enrofloxacin in red-eared sliders, a review of bacteria isolated from chelonians indicated that most had MIC values # 0.5 mg/ml. However, some bacteria such as Pseudomonas were resistant and had MIC values . 1.0 mg/ml. In the present study in loggerhead sea turtles, if we use a bacterium with an MIC value of 1 mg/ml as an example to determine the best dose to select, the Cmax/MIC ratio is 4.07 for group 1 turtles and 21.30 for group 2 turtles. From this pharmacodynamic parameter, the higher dose would be needed for a bacterium with MIC 5 1 mg/ml and should be efficacious for an organism with an MIC up to 2 mg/ml. In the mammalian literature, AUC0–24 is generally used in determining the AUC/MIC ratio, largely because of the popularity of once daily dosing of fluoroquinolones. When a computer algorithm is used,3 the AUC0–24 was 77.27 and 481.37 mg•hr/ml. With bacteria having MIC 5 1, the ratio would be 77.27 hr and 481.37 hr, respectively. With 100 to 125 used as the optimum ratio, 20 mg/kg of body weight should be efficacious against an organism with MIC 5 1 mg/ml. However, using AUC0–24 may not be appropriate because enrofloxacin seldom lasts in serum beyond 24 hr. If we use AUC0–`, both doses would result in ratios above 125. Still, plasma concentrations at 168 hr postadministration in the 20 mg/kg group was greater than eight times the plasma concentrations of the 10 mg/kg group. Because larger, less frequent doses may be more effective than smaller, more frequent ones,17 and no adverse effects were seen with either dosage rate in loggerhead sea turtles, a dosage rate of 20 mg/kg of body weight no more often than once per week is recommended until multiple dosage studies are conducted. Acknowledgments: We thank the staff of Clearwater Marine Aquarium for help in maintaining loggerhead sea turtles and collecting blood samples. Enrofloxacin assays were performed in the laboratory of Dr. Murray Brown, College of Veterinary Medicine, University of Florida. This project was supported by the Batchelor Foundation, College of Veterinary Medicine, University of Florida.

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