Koi (Cyprinus carpio) are popular companion animals in North America, and the demand for diagnosis and treatment of diseases in pet koi is increasing. Bacterial infections are often observed in sick koi as primary or secondary disease processes, but treatment can be difficult because sick fish often do not eat, making PO administration of antimicrobials unreliable, and IM administration requires handling, which often exacerbates stress and further suppresses the fish's immune system.
Danofloxacin is a synthetic fluoroquinolone antimicrobial developed specifically for veterinary use. Like all fluoroquinolones, it is bactericidal and exerts its antibacterial effects in a concentration-dependent manner by inhibiting bacterial DNA gyrase. Danofloxacin has a broad spectrum of activity against gram-positive bacteria, gram-negative bacteria, and Mycoplasma spp. The drug is available as a mesylate salt in an aqueous injectable form that can be administered IM or IV and as a soluble powder form that can be dissolved in drinking water.1 Danofloxacin is approved to treat specific bacterial infections in various food-producing animal species in the United States and Europe.1–3 Importantly, use of fluoroquinolones in food-producing animals is strictly regulated in many countries, and C carpio is considered to be a food-producing species of fish in many countries.
Danofloxacin is currently used to treat bacterial infections in koi and other pet and aquarium fish species because of the drug's low injection volume, lack of local tissue reaction in other species, wide antimicrobial spectrum in other species, and long dosing interval,1 and the authors frequently use danofloxacin to treat cutaneous bacterial infections and systemic septicemia in koi. However, although tissue depletion profiles of danofloxacin administered in medicated feed have been determined in several aquaculture species, including tilapia (Oreochromis mossambicus), sturgeon (Acipenser schrenckii), and sea bass (Dicentrarchus labrax),4–6 there is no information available on the pharmacokinetics of this drug in koi, and the currently recommended dosage of 10 mg/kg, IM, every 3 days is based on evidence extrapolated from cultured tilapia.4 Therefore, the purpose of the study reported here was to determine the pharmacokinetics and tissue concentrations of danofloxacin in koi following IM administration of a single dose.
Materials and Methods
The study protocol was approved by the Institutional Animal Care and Use Committee of the University of California-Davis.
Susceptibility of patient isolates to danofloxacin
The computerized medical records system of the University of California-Davis Veterinary Medical Teaching Hospital was searched to identify patient samples from ornamental fish that had been submitted for bacterial culture and antimicrobial susceptibility testing between 2010 and 2017. Aeromonas hydrophila, Aeromonas spp, Vibrio spp, and Pseudomonas spp isolates were tested by the broth microdilution methoda for susceptibility to danofloxacin in accordance with procedures recommended by the Clinical Laboratory Standards Institute.7 Briefly, bacterial isolates were incubated in brain-heart infusion broth for 4 to 6 hours at 35°C under aerobic conditions. Bacterial growth was added dropwise to saline (0.85% NaCl) solution to reach a McFarland standard of 0.5 as determined with a nephelometer. Ten microliters of this solution was then added to 11 mL of cation-adjusted Mueller-Hinton broth and 50 μL of the resulting mixture was used to inoculate wells containing danofloxacin at concentrations ranging from 0.12 to 1 μg/mL. Plates were incubated at 35°C for 16 to 24 hours under aerobic conditions, and minimum danofloxacin concentrations that inhibited 50% and 90% of isolates were determined. Quality control organisms that were used consisted of Staphylococcus aureus ATCC 29213, Enterococcus faecalis ATCC 29212, Escherichia coli ATCC 25922, and Pseudomonas aeruginosa ATCC 27853.8
Animals
Sixty-nine adult koi (Cyprinus carpio) weighing between 62 and 402 g were used in the study. Koi had been acquired from a commercial breederb and were kept as part of a research colony. All fish were considered healthy prior to inclusion in the study on the basis of results of a physical examination. They were housed together in a 980-L fiberglass tank with a direct flow-through freshwater system. A commercial pelleted diet designed for adult maintenance was offered once daily at a rate of 3% of the estimated biomass of the tank. Water temperature was measured daily and was maintained between 18°C and 19°C for at least 30 days prior to and during the study. Dissolved oxygen content of the tank water was measured weekly and was maintained at > 8 mg/L.
Experimental methods
Three randomly selected fish were designated as untreated negative controls, and the remaining 66 fish were randomly assigned to 11 treatment groups with 6 fish/group. All fish, including negative controls, were anesthetized via immersion in a bath of tricaine methanesulfonate (MS-222; 100 mg/L) buffered 1:1 (wt:wt) with sodium bicarbonate that was aerated with an air pump and air stone. When the fish had achieved a deep plane of sedation (ie, no response to a peduncle pinch), 2 fish from each group were randomly selected to undergo skin scraping and gill biopsy. Monogenean flatworms were identified in only 4 fish, and the level of parasitism was considered low enough to not warrant treatment or interfere with the present study.
Fish in the 11 treatment groups were weighed and given an injection of danofloxacin mesylatec at a dose of 10 mg/kg IM in the dorsal epaxial musculature to the left of midline at the level of insertion of the first dorsal fin ray; each fish was fin clipped to identify their treatment group. Fish were moved to a recovery bath immediately after fin clipping and were considered sufficiently recovered from anesthesia when they were swimming normally. Following recovery from anesthesia, fish were moved to holding tanks, with all fish from each group housed together in a single tank.
Fifteen, 30, and 45 minutes and 1, 4, 12, 24, 72, 96, 120, and 144 hours after administration of danofloxacin, fish in each of the treatment groups were anesthetized via immersion in a bath of tricaine methanesulfonate (100 mg/L) buffered 1:1 (wt:wt) with sodium bicarbonate, and a blood sample (0.5 to 0.7 mL) was collected from the caudal hemal arch with a 25-gauge needle and 1-mL syringe through a right lateral approach. Fish in the control group were anesthetized and sampled following fish from the 144-hour group. Blood samples were immediately transferred to evacuated tubes containing lithium heparin and mixed by inversion. Heparinized samples were centrifuged for 10 minutes at 1,500 × g within 1 hour after collection. Plasma was pipetted into sterile 1.5-mL microcentrifuge tubes and stored at −80°C until analyzed.
Following blood sample collection, fish were euthanized via immersion in a bath of tricaine methanesulfonate (500 mg/L) buffered 1:1 (wt:wt) with sodium bicarbonate for 10 minutes followed by pithing, in accordance with recommended euthanasia guidelines.9 A necropsy was performed on each fish, and samples of gill, liver, posterior kidney, anterior kidney, spleen, skin and muscle, and scales were collected and placed in sterile 1.5-mL microcentrifuge tubes. Tubes were stored at −80°C for later analysis. For fish euthanized 144 hours after danofloxacin administration, additional samples of each tissue type in addition to a 6-mm punch biopsy sample of the injection site (inclusive of skin and muscle) were obtained and preserved in neutral-buffered 10% formalin for histologic examination.
Plasma sample preparation
For analysis of plasma danofloxacin concentrations, plasma proteins were precipitated by diluting 200 μL of each plasma sample with 300 μL of a 9:1 (vol:vol) mixture of acetonitrile and 1M acetic acid containing ciprofloxacind (10 ng/mL) as an internal standard. Samples were vortexed for 2 minutes, refrigerated for 20 minutes, vortexed for an additional minute, and then centrifuged at 3,102 × g for 10 minutes at 4°C. Supernatant (150 μL) was transferred to an autosampler vial with insert, and 10 μL was used for measurement of plasma danofloxacin concentration by means of liquid chromatography–tandem mass spectrometry.
Tissue sample preparation
Approximately 10 to 910 mg of each tissue sample was weighed and placed in a 7-mL homogenizing vial.e Tissue samples were placed in a −20°C freezer for 10 minutes and were then homogenized twice at 5,500 rpm for 30 seconds in a tissue homogenizer,f with a 5-minute cooldown after each homogenization. Following homogenization, 200 to 1,000 μL of the homogenate was transferred to a microcentrifuge tube and centrifuged at 12,753 × g for 5 minutes. Supernatant (100 to 150 μL) was transferred to an autosampler vial with insert, and 10 μL was used for measurement of tissue danofloxacin concentration by means of liquid chromatography–tandem mass spectrometry.
Measurement of plasma and tissue danofloxacin concentrations
Plasma and tissue concentrations of danofloxacin were measured by means of liquid chromatography–tandem mass spectrometry with a turbulent-flow liquid chromatography system operated in laminar flow mode. Detection and quantification were conducted by means of selective reaction monitoring of the initial precursor ion for danofloxacin (m/z, 358) and the ciprofloxacin internal standard (m/z, 332). The responses for the product ions for danofloxacin (m/z, 245) and the ciprofloxacin internal standard (m/z, 205, 245, and 288) were plotted, and peaks at the proper retention times were integrated with standard software.g
Calibration curves were generated by means of linear regression to quantify danofloxacin concentration and were prepared fresh for each assay run. A weighting factor of 1/X was used for all calibration curves. For the plasma samples, danofloxacin calibration standards were prepared by dilution of the working standard solution to concentrations ranging from 2 to 25,000 ng/mL. For the tissue samples, calibration standards were prepared by diluting the working standard solution to concentrations ranging from 0.5 to 12,000 ng/mL in a 9:1 (v:v) mixture of acetonitrile and 1M acetic acid containing ciprofloxacind (10 ng/mL). In addition, quality control samples (2 concentrations within the standard curve) were included with each sample set as an additional check of accuracy. The extraction recovery for each tissue type was determined by spiking drug-free fish tissues with danofloxacin. These tissues were processed in the same manner as described for the study samples. Extraction efficiency (ie, percentage recovery) was 106% for plasma samples, 108% for gill samples, 86% for liver samples, 117% for posterior kidney samples, 103% for anterior kidney samples, 92% for spleen samples, 107% for skin and muscle samples, and 97% for samples of the scales.
The assay response was linear, with correlation coefficients ≥ 0.99. Intra- and interday precision and accuracy of the assay were determined by assaying replicates (n = 6) of quality control samples. Precision and accuracy values were within 7% and 11%, respectively, of the nominal concentrations. The technique was optimized to provide a limit of quantitation of 2 ng/mL for plasma samples and a limit of quantitation ranging from 0.09 to 0.1 ng/mg for tissue samples. The limit of detection was 1 ng/mL for plasma samples and 0.05 to 0.06 ng/mg for tissue samples.
Pharmacokinetic analysis
Pharmacokinetic parameters were calculated by means of noncompartmental analysis of sparse data, with plasma drug concentrations from all fish analyzed simultaneously in a way that enabled estimation of SEs of the mean for maximum plasma concentration (Cmax) and area under the concentration-versustime curve from time 0 to the last measured concentration (AUC0–last). Standard errors of the mean were calculated as described by Nedelman and Jia,10 with the modification described by Holder.11
Histologic examination of tissue samples
Paraffin-embedded tissues were cut into 5-μm-thick thick sections and stained with H&E stain. All tissue slides were evaluated by one of the authors (WCS) and a collaborating veterinary pathologist.
Results
All fish maintained normal feed intake and demonstrated normal behavior after administration of danofloxacin.
Susceptibility of patient isolates to danofloxacin
Fifty-one A hydrophila isolates, 31 other Aeromonas isolates, 25 Vibrio isolates, and 13 Pseudomonas isolates were available for testing susceptibility to danofloxacin. Minimum danofloxacin concentrations that inhibited growth of 50% of the isolates were 0.25 μg/mL for the other Aeromonas isolates and the Vibrio isolates and 0.5 μg/mL for the A hydrophila and Pseudomonas isolates. The minimum danofloxacin concentration that inhibited growth of 90% of the isolates was 1.0 μg/mL for all 4 groups of isolates. For all 4 quality control strains, minimum inhibitory concentrations were within expected ranges (S aureus ATCC 29213, 0.12 to 0.25 μg/mL; E faecalis ATCC 29212, 0.5 to 1.0 μg/mL; E coli ATCC 25922, < 0.06 μg/mL; and P aeruginosa ATCC 27853, 0.5 to 1.0 μg/mL).
Plasma danofloxacin concentrations
Plasma danofloxacin concentrations were quantifiable in all 6 fish euthanized 15 minutes after drug administration and in 2 of the 6 fish euthanized 144 hours after drug administration (Table 1).
Plasma pharmacokinetic properties of danofloxacin following IM administration of a single dose (10 mg/kg) in koi (Cyprinus carpio).
| Variable | Value |
|---|---|
| λz (h−1) | 0.046 |
| t1/2λ (h) | 15.0 |
| tmax (h) | 0.75 |
| Cmax (ng/mL)* | 8,315.7 ± 1,506.5 |
| tlast (h) | 144 |
| Clast (ng/mL) | 6.08 |
| AUC0–last (h·ng/mL)* | 249,717 ± 40,367 |
| AUC0–∞ (h·ng/mL) | 249,848 |
| Vz/F (mL/kg) | 866.43 |
| Cl/F (mL/kg/h) | 40.0 |
Data represent mean ± SE.
AUC0–last = Area under the plasma concentration-versus-time curve from time 0 to the last measured concentration. AUC0–∞ = Area under the plasma concentration-versus-time curve from time 0 extrapolated to infinity. Clast = Plasma concentration at tlast. Cl/F = Apparent plasma clearance. Cmax = Maximum plasma concentration. λz = Terminal elimination rate constant. t1/2λ = Terminal half-life. tlast = Last observation time at which danofloxacin was detected in plasma. tmax = Time to maximum plasma concentration. Vz/F = Apparent volume of distribution during the terminal phase.
Tissue danofloxacin concentrations
Danofloxacin was detected in all examined tissues from all 6 fish euthanized 15 minutes after drug administration and was detected in some tissues from 3 of the 6 fish euthanized 144 hours after drug administration. There was a distinctly bimodal distribution of danofloxacin in the examined tissues (Table 2), and concentrations varied widely among individual fish.
Mean ± SD tissue concentrations of danofloxacin (ng/g)* following IM administration of a single dose (10 mg/kg) to koi.
| Time after injection | Scales | Gills | Posterior kidney | Anterior kidney | Spleen | Liver | Skin and muscle |
|---|---|---|---|---|---|---|---|
| 15 min | 0.44 ± 0.28 | 2.01 ± 1.17 | 4.13 ± 2.08 | 5.53 ± 1.97 | 5.30 ± 5.75 | 1.88 ± 0.77 | 0.21 ± 0.13 |
| 30 min | 1.69 ± 3.66 | 15.82 ± 32.54 | 24.44 ± 50.49 | 34.39 ± 70.49 | 9.14 ± 11.01 | 11.59 ± 25.30 | 1.54 ± 3.04 |
| 45 min | 52.90 ± 94.60 | 24.17 ± 26.50 | 194.64 ± 397.64 | 66.13 ± 88.24 | 25.29 ± 19.87 | 58.42 ± 103.37 | 5.73 ± 7.30 |
| 1 h | 5.51 ± 8.39 | 6.96 ± 8.72 | 14.41 ± 19.67 | 26.33 ± 32.0 | 10.83 ± 12.15 | 7.24 ± 7.67 | 1.19 ± 1.14 |
| 4 h | 0.87 ± 0.40 | 5.76 ± 4.14 | 18.0 ± 8.93 | 16.98 ± 10.25 | 5.57 ± 2.81 | 8.85 ± 6.91 | 1.64 ± 1.03 |
| 12 h | 27.90 ± 28.86 | 31.32 ± 19.85 | 69.05 ± 35.80 | 96.59 ± 68.57 | 59.74 ± 37.47 | 23.62 ± 12.19 | 11.64 ± 7.11 |
| 24 h | 21.87 ± 23.68 | 7.96 ± 5.64 | 47.89 ± 38.83 | 66.38 ± 47.04 | 45.12 ± 51.04 | 14.47 ± 12.38 | 7.97 ± 7.35 |
| 72 h | 3.59 ± 2.24 | 7.93 ± 6.09 | 11.47 ± 7.41 | 26.95 ± 20.22 | 10.36 ± 12.75 | 3.57 ± 2.19 | 2.65 ± 2.23 |
| 96 h | 0.50 ± 0.37 | 4.73 ± 4.67 | 15.44 ± 15.26 | 44.96 ± 41.56 | 15.12 ± 18.23 | 4.59 ± 3.92 | 4.02 ± 3.42 |
| 120 h | 0.37 ± 0.60 | 0.82 ± 1.31 | 2.41 ± 3.69 | 4.50 ± 8.41 | 1.35 ± 2.13 | 1.02 ± 1.82 | 0.54 ± 1.08 |
| 144 h | 0.02 ± 0.02 | 0.016 ± 0.007 | 0.13 ± 0.009 | 0.10 ± 0.04 | 0.05 ± 0.02 | 0.006 ± 0.001 | 0.01 ± 0.005 |
Tissue concentrations have not been corrected for extraction efficiency (ie, percentage recovery).
Histologic evaluation
No histologic abnormalities were seen in skin and muscle biopsy samples obtained from injection sites 144 hours after drug administration. Specifically, no evidence of myodegeneration, inflammation, or hematoma or seroma formation was seen. Similarly, no histologic abnormalities were seen in any of the other tissue samples that were examined.
Discussion
Results of the present study suggested that in koi, IM administration of danofloxacin at a dose of 10 mg/kg resulted in detectable danofloxacin concentrations in plasma and tissues within 15 minutes after drug administration. In addition, mean danofloxacin concentrations in gill, liver, posterior kidney, anterior kidney, spleen, and skin and muscle samples were higher than the concentration that inhibited 90% of patient A hydrophila, Aeromonas spp, Vibrio spp, and Pseudomonas spp isolates for at least 96 hours after drug administration. Unfortunately, these concentrations derived from tissue samples cannot be used to predict antibacterial efficacy, and controlled studies are needed to determine whether danofloxacin is efficacious against common aquatic pathogens. In mammals, the efficacy of fluoroquinolone drugs can often be predicted on the basis of the ratio of maximum observed concentration to minimum inhibitory concentration, with a ratio ≥ 10 generally indicative of efficacy.12 However, similar data do not exist for fish; therefore, predicting efficacy on the basis of this ratio in fish may not be useful. Further studies investigating the pharmacodynamics of danofloxacin in koi are warranted to provide better insight on the efficacy of using danofloxacin to treat bacterial infections in fish.
In all tissues in the present study, there was a distinct bimodal distribution of drug concentration with peaks between 30 and 45 minutes and again between 12 and 24 hours after danofloxacin administration. There was also wide individual variation in tissue drug concentration at all time points. Similar marked individual variation has been observed with IM administration of alfaxalone in koi.13 There may be variability in the absorption of drugs following IM injection in teleost fish because of the unique cellular and structural organization of their tissues and the fact that they have at least 2 distinct types of skeletal muscle.14 Differential absorption of danofloxacin from red versus white muscle may have accounted for the bimodal distribution of drug concentrations in tissues. Also, in the present study, we attempted to standardize the injection site and procedure; however, the depth of injection was not standardized because of variability in the body size of fish included in the study. As a result, there may have been variability in the fraction of drug injected into red and white muscle types, which could have accounted for some of the individual variability in drug concentrations among fish.
Another potential explanation for the variability in drug concentration between individual fish in the present study could be the structural organization of muscle tissue in teleost fish. In teleost fish, muscle tissues have a lower tolerance for injection of large volumes of medication than is the case for muscle tissue in mammals or birds, and drug leakage from the injection site is not uncommon in fish. A study15 of the pharmacokinetics of meloxicam in Nile tilapia (Oreochromis niloticus) showed variation in absorption of drug following IM injection that was attributed to the structure of teleost muscle.
All injections were administered in the same site on all fish in the present study to avoid potential complications of drug metabolism associated with the renal portal system and to ensure uniformity of muscle biopsy samples. The lack of inflammation or myodegeneration at the site of injection in all fish suggested that danofloxacin is safe to administer without dilution of the stock aqueous formulation. The absence of seroma or hematoma formation reduced the possibility of an iatrogenic depot effect that can be seen in other animals following IM administration of various compounds.
In the present study, danofloxacin was rapidly absorbed after IM injection in koi, with maximum plasma concentration reached approximately 45 minutes after drug administration. In addition, the drug could still be detected in some tissues 144 hours after administration. Danofloxacin was distributed throughout all tissues examined (gill, spleen, liver, anterior kidney, posterior kidney, skin and muscle, and scales) and achieved mean concentrations exceeding the minimum concentration necessary to inhibit the growth of 90% of patient isolates within 30 minutes after administration in all tissues. Because protein binding was not measured in the present study, it is not possible to make predictions on the basis of pharmacokinetic-pharmacodynamic relationships. However, none of the fish in our study had any apparent adverse reactions to treatment with danofloxacin, and histologic evaluation of tissues did not reveal evidence of any abnormalities associated with treatment. Further study is warranted to determine the efficacy of danofloxacin against bacterial infections in koi. Our findings suggested that IM administration of danofloxacin at a dose of 10 mg/kg resulted in detectable concentrations in all tissues examined. However, dosing and dosing interval recommendations for danofloxacin should be based on susceptibility of the isolated pathogen, empirical evidence, and any legal limitations surrounding its use.
Acknowledgments
No third-party funding or support was received in connection with this study or the writing or publication of the manuscript. The authors declare that there were no conflicts of interest.
At the time of the study, Dr. Parker-Graham was receiving a University of California-Davis Aquatic Animal Health Fellowship supported by the California Academy of Science Steinhart Aquarium, Monterey Bay Aquarium, and Hubbs-Sea World Research Institute.
The authors thank Matthew F. Sheley of the California Animal Health and Food Safety Lab System for collaboration and help.
Footnotes
Sensititre BOPO6F Vet AST plate, Thermo Fisher Scientific Inc, Waltham, Mass.
Blackwater Creek Koi Farm, Eustis, Fla.
Advocin (180 mg/mL), Zoetis, Parsippany, NJ.
Sigma Aldrich, St Louis, Mo.
Homogenizing vial, Omni International, Kennesaw, Ga.
Bertin Instruments, Rockville, Md.
Thermo Fisher Scientific Inc, San Jose, Calif.
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