Antibiotic uptake by cultured Atlantic cod leucocytes and effect on intracellular Francisella noatunensis subsp. noatunensis replication.

The granuloma disease caused by Francisella noatunensis subsp. noatunensis in farmed Atlantic cod has not been successfully treated by use of antibacterials, even when antibacterial resistance testing indicates a sufficient effect. The reason for this treatment failure may be the intracellular existence of the bacteria within immune cells, mainly macrophages. To investigate the effect of antibacterials on intracellular Francisella replication, we established a protocol for the detection of drugs within Atlantic cod immune cells using high-performance liquid chromatography (HPLC). When the uptake and intracellular concentrations of oxolinic acid and flumequine were analysed in isolated adherent head kidney leucocytes (HKLs) by HPLC, we found that uptake was rapid and the intracellular concentrations reflected the extracellular exposure concentrations. To investigate the effect of the antibacterial compounds on intracellular bacterial replication, adherent HKLs experimentally infected with the bacteria were analysed using flow cytometry and intracellular labelling of bacteria by specific antibodies. We found that flumequine did not inhibit intracellular bacterial replication. Unexpectedly, the results indicated that the intracellularly effiacy of the drug was reduced. The HPLC method used proved to be highly applicable for accurate determination of intracellular drug concentrations. When combined with sensitive and specific flow cytometry analyses for identification and measurement of intracellular bacterial replication, we suggest that this approach can be very valuable for the design of antibacterial treatments of intracellular pathogens.

Anesthesia induces stress in Atlantic salmon (Salmo salar), Atlantic cod (Gadus morhua) and Atlantic halibut (Hippoglossus hippoglossus).

Stress in response to anesthesia with benzocaine, MS-222, metomidate and isoeugenol was studied in Atlantic salmon (Salmo salar), Atlantic halibut (Hippoglossus hippoglossus), and Atlantic cod (Gadus morhua) with no concomitant stress from handling or confinement in association with anesthesia or sampling. All of the anesthetics tested induced a stress response in all species, displayed by a release of cortisol to the water. MS-222 anesthesia elicited the highest cortisol release rates, reaching maximum levels 0.5 h post-exposure and returning to basal levels after 3-4 h. Benzocaine anesthesia caused a bimodal response where the initial peak in cortisol release rate was followed by a second increase lasting towards the end of the trial (6 h). This bimodality was more profound in Atlantic salmon than in Atlantic halibut and Atlantic cod. Metomidate anesthesia induced the lowest release of cortisol of the agents tested in both Atlantic halibut and Atlantic cod, but resulted in a bimodal response in Atlantic salmon where the initial increase in cortisol release was followed by a larger increase peaking at 2-2.5 h post exposure before returning to basal levels after 5 h. The stress induced in Atlantic salmon by isoeugenol anesthesia resembled that of MS-222, but did not reach the same elevated level. Overall, the cortisol release was most profound in Atlantic salmon followed by Atlantic halibut and Atlantic cod.

Efficacy of orally administered flumequine in the treatment of vibriosis caused by Listonella anguillarum in atlantic cod Gadus morhua.

The efficacy of orally administered flumequine in the treatment of experimentally induced vibriosis in Atlantic cod Gadus morhua was investigated. Cod (mean +/- SD, 120 +/- 30 g) were randomly distributed to twelve tanks and bath challenged for 1 h with Listonella anguillarum serotype O2alpha, strain HI-610, using a dose of 9.2 x 10(6) CFU ml(-1). At 3 d post-challenge, medication was introduced in 10 of the groups at doses of 2.5, 5, 10, 15 and 25 mg flumequine kg(-1) body weight d(-1) in duplicate. The medication was administered on Days 1, 2, 4, 6, 8, and 10 after the initiation of treatment. In challenged unmedicated fish, mortality started on Day 4 post-challenge, reaching a final cumulative mortality of 82% at Day 18. In the medicated groups, mortality started on Days 3 to 5 post-challenge, reaching final cumulative mortalities of 42, 49, 37, 37 and 23% respectively for the fish treated with 2.5, 5, 10, 15 and 25 mg flumequine kg(-1) body weight d(-1). Survival of medicated fish in all groups was significantly greater than in challenged unmedicated fish (p < 0.001). Twenty-four h following the final medication, HPLC analysis found a linear relationship between doses and mean concentrations of the drug in plasma, muscle and liver.

One-step liquid chromatographic method for the determination of oxytetracycline in fish muscle.

A one-step simple and rapid high performance liquid chromatography (HPLC) method was developed for the determination of oxytetracycline (OTC) in fish tissue. The method involves liquid extraction of muscle tissue, precipitation of proteins and reversed phase HPLC analysis with spectrophotometric detection. The limit of quantitation of OTC in spiked fish muscle was 0.04 microg/g and the method showed high linearity (r(2) = >0.999) in the working range of 0.04-2 microg/g. The precision (%R.S.D.) was between 1.9 and 7.5% for the concentration range 0.04-1.0 microg/g and there was no significant difference between the concentrations determined on three different test days for all four spiked concentrations. The percentage recovery over the spiked concentration range 0.04-1.0 microg/g was consistently within a narrow range of 33-35%. While the method had the advantage of high precision, sensitivity and linearity, the method's additional salient advantages included high sample through-put (60 individual preparations per day) and minimum amount of consumables, time and labour required to perform the analysis. The method was successfully applied to a pharmacokinetic study.

Pharmacokinetics of florfenicol in cod Gadus morhua and in vitro antibacterial activity against Vibrio anguillarum.

The pharmacokinetic profile of the antibacterial agent florfenicol was studied in plasma after intravenous (i.v.) injection and in plasma, muscle and liver following oral (p.o.) administration to cod Gadus morhua, held in seawater at 8 degrees C and weighing 100 to 200 g. Following i.v. injection, the plasma drug concentration-time profile showed 2 distinct phases. The plasma distribution half-life (t1/2alpha) was estimated to be 1.6 h, the elimination half-life (t1/2beta) to be 43 h, the total body clearance (ClT) to be 0.015 1 kg(-1) h(-1) and mean residence time (MRT) to be 74 h. The volume of distribution at steady state, Vd(ss), was calculated to be 1.1 l kg(-1). Following p.o. administration, the bioavailability was estimated to be 91%, the peak plasma concentrations (Cmax) to be 10.8 microg ml(-1) and the time to peak plasma concentrations (Tmax) to be 7 h. Corresponding Cmax and Tmax values were 13.0 microg g(-1) and 9 h, respectively, in muscle and 12.1 microg g(-1) and 9 h, respectively, in liver. The in vitro minimum inhibitory concentration (MIC) values of florfenicol against 3 Vibrio anguillarum strains isolated from diseased cod (A-21, HI-610, HI-618) were 0.5 microg ml(-1) for all 3 strains.

The stability and persistence of diflubenzuron in marine sediments studied under laboratory conditions and the dispersion to the sediment under a fish farm following medication.

A high performance liquid chromatographic (HPLC) method was developed to determine the concentration of diflubenzuron, a delousing agent used in fish farming, in marine mud and shell sand. The recovery of diflubenzuron from mud was 100.8+/-1.1% and 105.5+/-4.3% for shell sand. The limit of quantitation was found to be 0.1 microg g(-1). The stability of diflubenzuron was studied under laboratory conditions in marine sediments at different temperatures (4 and 14 degrees C). No degradation of diflubenzuron occurred in the organic rich mud sediment or in the shell sand sediment during the experimental period of 204 days. Increasing the temperature from 4 to 14 degrees C had no effect on the stability. Furthermore, diflubenzuron showed to be persistent in both mud and shell sand sediment since no detectable diffusion from the sediment to the water phase occurred during the experimental period of 204 days. Increasing the water current in the tanks had no effect on the persistence. Under field conditions, the concentrations of diflubenzuron found in the organic material from sediment traps placed 2 m from the bottom under the cage in a fish farm during medication were high and ranged from 71 to 259 microg g(-1). The concentrations of diflubenzuron in the sediment under the fish farm were, however, low, with a maximum concentration of 5.4 microg g(-1). The dispersion of diflubenzuron to the sediment was limited to less than 20 m from the edge of the cage in every direction. Fifteen months following the medication, only traces (< 0.1 microg g(-1)) of diflubenzuron were detected in the sediment under the fish farm. Possible explanations for this decrease are resuspension and redistribution of sediment and/or oxic degradation of the drug.