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Enrofloxacin (E) is a commonly used antimicrobial in reptile medicine. However, few studies have been performed in reptile species to determine therapeutic blood levels of the fluoroquinolone (FQ) class of antimicrobial agents. The aim of the present study was to evaluate the pharmacokinetics of E and its metabolite ciprofloxacin (C) in 21 yellow-bellied slider turtles (Trachemys scripta scripta). The animals were administered 10 mg/kg of E via intracoelomic injection. Blood was collected at scheduled times and analyzed using a validated high-performance liquid chromatography (HPLC) fluorescence method. The turtles showed some indications of transient pain following administration. The pharmacokinetics of E and C were long lasting (196 hours and 168 hours, respectively). This could be of concern where turtles are used for food because of drug tissue residues. The administered dose seemed to be optimal for clinical application because (1) drug concentrations in blood were appropriate for treating bacterial infections without adversely affecting the patient and (2) effective maximum concentration/minimum inhibitory concentration (Cmax/MIC) and area under the simulated blood-time curve (AUC)/MIC ratios were achieved. In conclusion, the dose of 10 mg/kg administered via intracoelomic injection in yellow-bellied slider turtles appeared safe and achieved optimal pharmacodynamic parameters.