Pharmacokinetic and efficacy studies on bath-administering potentiated sulphonamides in Atlantic...

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Efficacy of Bath-AdministeredFlumequine and Oxolinic Acid inthe Treatment of Vibriosis in SmallAtlantic HalibutOle B. Samuelsen aa Department of Clinical Biology, Section of Pharmacology,Armauer Hansens Hus, 5021, Bergen, Norway

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To cite this article: Ole B. Samuelsen (1997): Efficacy of Bath-Administered Flumequineand Oxolinic Acid in the Treatment of Vibriosis in Small Atlantic Halibut, Journal of AquaticAnimal Health, 9:2, 127-131

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Journal of Aquatic Animal Health 9 :127-131, 1997© Copyright by the American Fisheries Society 1997

Efficacy of Bath-Administered Flumequine andOxolinic Acid in the Treatment of Vibriosis in

Small Atlantic Halibut

OLE B . SAMUELSENDepartment of Clinical Biology, Section of Pharmacology

Armauer Hansens Ifus, 5021, Bergen, Norway

Abstract .-In this investigation, the efficacies of bath-administered flumequine and oxolinic acidin the treatment of vibriosis in small Atlantic halibut Hippoglossus hippoglossus were examined .In a laboratory challenge experiment, halibut (3-5 g) were exposed to Vibrio anguillarum strain(isolate HI 11347) at a concentration of 4.0 X 10 8 bacteria/mL for 30 min. Bath treatments,initiated 24 h after challenge at a concentration of 150 mg of flumequine or 200 mg of oxolinicacid per liter for 72 h, significantly (P < 0.05) lowered the V. anguillarum-specific mortality inthe drug-treated groups compared with the untreated control group. In another trial in which thebacterial challenge was reduced to 3.2 X 108 bacterialmL (80% of initial dose) and the bathtreatment with flumequine (150 mg/mL) was begun on days 2, 3, or 5 postchallenge, the cumulativemortality was significantly (P < 0.05) different from the control group only when treatment wasinitiated on day 2 postchallenge . Bacterial examination of kidneys from dead and surviving fishindicated the pathogen was quickly and effectively eliminated by the antibacterials .

Flumequine (FLU) and oxolinic acid (OXA) arebroad-spectrum synthetic antibacterial agents ofthe 4-quinolone series . Quinolones are especiallyactive against gram-negative bacteria, and the an-tibacterial activity is based on the inhibition ofDNA-gyrase, resulting in an unstable condensationof the DNA configuration of the bacterial DNAmolecule during cell division (Wolfson and Hoop-er 1989). In vitro studies have shown potent ac-tivity of both FLU and OXA against several bac-teria pathogenic to fish (Stamm 1989 ; Hoie et al .1992 ; Martinsen et al . 1992), and pharmacokineticstudies have shown the drugs to be well distributedin fish (Sohlberg et al . 1990 ; Martinsen and HOTS-

berg 1995). Therefore, FLU and OXA have beenused extensively to treat a number of systemic bac-terial infections in fish (Michel et al . 1980 ; Austinet al . 1983 ; Rodgers and Austin 1983 ; Scallan andSmith 1985 ; O'Grady et al . 1988). In recent years,bacterial strains highly resistant to these antibac-terials have also been reported (Aoki et al . 1985 ;Barnes et al . 1990 ; Høie et al . 1992 ; Martinsen etal . 1992).

Injection into the peritoneal cavity or musclerepresents the most effective and accurate meansof administering a therapeutic dose of an antibac-terial agent to an individual fish . Individual injec-tion, however, is inappropriate as the routine meth-od of chemotherapy due to major costs in time andlabor; at present it is only used in the treatment of

valuable parent fish . Oral administration of drugswith food pellets containing the desired antibac-

terial substance permits treatment of large quan-tities of fish relatively easily and has become theprime route of medication . However, diseased fishtend to be anorexic, thus oral administration willinevitably result in uneven distribution of the an-tibacterial in the stock. As a result, only the healthyindividuals that are still feeding are protected bythe antibacterial . Administration of antibacterialagents via bath treatment is independent of thefeeding behavior of the fish, and bath treatmentmay also be an alternative for treating smaller fish .It has been demonstrated that bath administrationof erythromycin with a surfactant could eliminateAeromonas salmonicida from the intestine of fish(Roberts 1980), whereas administration ofFLU viabath treatment has been demonstrated under lab-oratory conditions to eliminate or provide controlof stress-inducible furunculosis infections insmolts of Atlantic salmon Salmo salar (O'Gradyand Smith 1992) . Bath administration of FLU andOXA has shown the drugs to be well distributedin tissues of small Atlantic halibut Hippoglossushippoglossus (Samuelsen and Lunestad 1996) .

The purpose of this study was to evaluate theefficacy of administrating FLU and OXA by bathto treat a laboratory-induced Vibrio anguillaruminfection in small Atlantic halibut.

Methods

Chemicals and Fish

The FLU and OXA were obtained from NorskMedisinaldepot (Bergen, Norway) and sodium hy-

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droxide (NaOH; pro analysi grade) was obtainedfrom Merck (Darmstadt, Germany) .Approximately 250 Atlantic halibut, obtained

from Tinfos Aqua (Kvinesdal, Norway) and vary-ing in weight from 3 to 5 g, were maintained in acircular (1 m deep X 2.5 m, inside diameter) flow-through seawater storage tank at the laboratory ofthe Institute of Marine Research (Bergen, Nor-way) . The seawater had a salinity of 33%o and wasmaintained at 12.0 ±- 0.5°C . The fish were fed drypellets (Ewos Aqua A/S, Skårer, Norway) and wereactively feeding prior to challenge.

ChallengeThe bacterial strain V. anguillarum serotypeO2a (isolate HI 11347) provided by the Institute

of Marine Research was used for the challengeexperiments . This strain is pathogenic to halibutand susceptible to OXA andFLU, with an in vitrominimum inhibitory concentration (MIC) of 0.015

ug/mL for both compounds (Samuelsen and Lu-nestad 1996). Fish from the storage tank were ran-domly assigned to seven groups of 15 fish each .Waterborne challenge experiments were per-formed by exposing groups 1, 2, and 3 to 4 .0 X108 bacteria/mL for 30 min (trial 1) and groups 4,5, 6, and 7 to 3.2 X 108 bacteria/mL for 30 min(trial 2) ; fish were then transferred to flow-throughseawater tanks. The reduction in bacterial chal-lenge density in trial 2 was based on the resultsfrom trial 1 confirming a high mortality rate duringthe first 2 d after challenge. Bacterial density wasdetermined by plate counts and optical density

measurements.

Bath TreatmentBath treatments were carried out in separate

40-L glass aquaria, each containing 20 L of oneof the drug solutions. Flumequine and OXA wereeach dissolved in 50 mL of 0.025 M NaOH, son-icated for 5 min, and added to the seawater 1 dprior to initiating bath treatment to obtain com-plete dissolution of the drugs. This operation in-creased the pH of the bath solution from 8.0 to8 .1 . According to the procedure of Samuelsen andLunestad (1996), bath concentrations of 150 mgFLU/L and 200 mg OXA/L were used, and the fishwere kept in the baths for 72 h . This treatmentregime maintained muscle levels in excess of aMIC of 0.0625 ug/mL for approximately 7 d forFLU and 8 d for OXA. The bath solutions wereaerated and the aquaria were equipped with aquar-ium filter pumps to filter off fecal particles . Theconcentrations of FLU and OXA in the baths were

analysed by high-performance liquid chromatog-raphy (HPLC) before and after bath treatment ac-cording to the procedure of Lunestad et al. (1995) .

Trial 1 .-Twenty-four hours after challenge thefish from group 1 were transferred to an aquariumcontaining OXA, and fish from group 2 were trans-ferred to an aquarium containing FLU. The fishwere kept in the drug solutions for 72 h; at whichtime the surviving fish were transferred to theflow-through seawater tanks. The fish in group 3received no medication and served as controls .

Trial 2.-For the fish in groups 4, 5, and 6,treatment with FLU was initiated at 2, 3, and 5 dafter challenge, respectively, and continued for 72h. The fish in group 7 received no medication andserved as controls .The fish in both trials were observed for 21 d

after challenge, and deaths were recorded daily.An additional unchallenged, unmedicated group of50 fish was used to determine the natural mortalityin the population . Mortality data were analyzed bythe chi-square test .

Detection of PathogenKidney samples from dead and surviving fish

from both trials were examined for the presenceof V. anguillarum by bacterial cultivation . Beforenecropsy, the Atlantic halibut were disinfectedwith 70% ethanol . Kidney samples were inoculat-ed on nutrient agar (Oxide, Basingstoke, UK) sup-plemented with 5% defibrinated sheep blood and1 .5% NaCl . The agar plates were incubated aerob-ically at 15°C for a maximum of 7 d. Bacterialisolates were tested for sensitivity to the vibrios-tatic agent 0/129 and biochemical reactions in theAPI 20E system (bio Mérieux, Marcy-l'Etoile,France).

ResultsThe measured drug concentrations in the baths

were all within the limit of ±0.5% of the assignedconcentration . The decreases in drug concentrationin all baths during treatment were less than 0.5% .No mortality was observed in the unchallengedpopulation .

Trial 1The first deaths occurred on day 1 following

challenge (Figure 1) . Among challenged controls(group 3), the cumulative mortality rose rapidly to80% and then plateaued. At day 21, the final cu-mulative mortality was 93%. Mortality of fishtreated with OXA (group 1) and FLU (group 2)plateaued at 6% at days 2 and 1 postchallenge,

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respectively . A final cumulative mortality of 13%was recorded for both groups . All deaths occurredduring the first 7 d after challenge. A significantlyhigher mortality rate was observed for challengedunmedicated control fish (P < 0.005) comparedwith unchallenged fish . Survival of medicated fishwas significantly (P < 0.05) greater than survivalof challenged unmedicated fish .The pathogen was isolated from the kidneys of

dead fish from all groups on day 1 and from group3 on days 2, 4, and 7 postchallenge . The pathogenwas not isolated from the survivors from any ofthe groups (1, 2, and 3) or from the dead fish fromgroup 1 on day 5 or from group 2 on day 7 post-challenge.

Trial 2In trial 2, the first deaths occurred on day 2

postchallenge (Figure 2) . During the first 4 d, cu-mulative mortality rose to 66% among unmedi-cated controls (group 7) . At day 21 the final cu-mulative mortality was 80%. For fish medicatedafter 2, 3, and 5 days (groups 4, 5, and 6), finalcumulative mortalities were 46%, 60%, and 66%,respectively . All deaths occurred during the first7 d after challenge. A significantly higher mortal-

ity rate was observed for challenged unmedicatedcontrol fish (P < 0.005) compared with unchal-lenged fish, and survival of fish medicated after 2d (group 4) was significantly (P < 0.05) greaterthan survival of unmedicated fish (group 7) .

The pathogen was not isolated from the survi-vors in any of the medicated groups . Neither wasit isolated from the dead fish in the group thatbegan medication 2 d after challenge and werecultured at day 4 nor from the group that beganmedication 5 d after challenge and were culturedat days 7 and 8. A reduction in the number of V.anguillarum-specific colonies formed, as judgedby a change from confluent growth to separatedcolonies, was observed in both groups 4 and 5 1d after treatment began .

Discussion

Designing an effective dose regime for an an-tibacterial agent requires knowledge of both thepharmacokinetic properties of the drugs and thesensitivity of the pathogen to the agents . It hasbeen shown that the V. anguillarum isolate HI11347 is susceptible to FLU and OXA with a MICof 0.015 ug/mL for both drugs and that musclelevels of FLU andOXA greater than the MIC were

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maintained for 7 and 8 d, respectively, in muscleof small Atlantic halibut in a treatment regimeidentical to the regime used in this investigation(Samuelsen and Lunestad 1996). Peak muscle con-centrations of 14 .2 ug/g for FLU and 9.4 ug/g forOXA were achieved (Samuelsen and Lunestad1996) . Therefore, the low MICs and high tissueconcentrations make these drugs good candidatesfor treating vibriosis in this species. The resultsobtained in this investigation support this potentialuse of FLU and OXA .

Trial 1 demonstrated that bath treatment withFLU and OXA against experimentally inducedvibriosis successfully reduced V. anguillarum-spe-cific mortalities in the medicated groups comparedwith the unmedicated group. Although no signif-icant differences in mortality (P > 0.05) occurredbetween groups in which medication began 3 or 5d after challenge (groups 5 and 6) and unmedicatedcontrols (group 7) in trial 2, a trend of increasingefficacy was observed with decreasing time be-tween challenge and initiation of treatment.Data from efficacy studies and research in the

use of antibacterials to treat infections in Atlantichalibut are very limited . However, the experimen-tal design used in trial 1 of this study was identicalto the design of a previous study examining the

efficacy of a bath-administered combination of sul-phadimidine (500 ug/mL) and trimethoprim (100ug/mL) to treat vibriosis in small halibut. It is,therefore, legitimate to compare these two sets ofdata . A cumulative mortality of 40% was recordedin the group treated with the sulphadimidine-tri-methoprim combination compared with 93% cu-mulative mortality in the challenged unnmedicatedcontrol group (O . B . Samuelsen, unpublished re-sults) . The MIC for the sulphadimidine-trimeth-oprim combination against the V. anguillarumstrain HI 11347 was 0.8 ug/mL (Samuelsen, un-published results) . The therapeutic efficacy ob-tained in trial 1 in this investigation is similar tothe efficacy obtained by administering the quin-olones OXA and sarafloxacin orally to small turbotScophthalmus maximus via bioencapsulation in Ar-temia fransciscana (Duis et al . 1995). Duis et al .(1995) reported a mean mortality rate of 6% in theOXA-treated groups and 14 .1 % in the sarafloxa-cin-treated groups compared with a75% mortalityin the unmedicated control group. Medication was,however, initiated immediately after challenge.Bacterial cultivation of kidney samples from deadand surviving fish from both trials in the presentstudy, although based on few samples, indicatedthat the pathogen was effectively and quickly elim-

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inated from the fish by application of either FLUor OXA.

In conclusion, this investigation shows that bathtreatments with FLU or OXA effectively treated alaboratory-induced infection of V. anguillarum insmall Atlantic halibut . The importance of rapidinitiation of treatment was demonstrated in trial 2 .Because administration of antibacterials via bathis independent of the feeding behavior of the fish,this route may be advantageous in treating vibri-osis because one of the first signs of this diseaseis loss of appetite (Hjeltnes and Roberts 1993) .Bath treatment is easy to carry out, and a largenumber of fish can be treated in a comparativelysmall volume of water. However, environmentalconsiderations and the large amount of drug need-ed for one treatment make this technique unsuit-able for use in sea cages .

Acknowledgments

This work was funded by the Norwegian Re-search Council . The technical assistance of KariAndersen, Audun Hoylandkjxr, and Hari Rudra ishighly appreciated . I also thank Odd Magne Rod-seth at the Institute of Marine Research for pro-viding the bacterial strain and Ronald R . Schelinefor critical reading of the manuscript .

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