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Uptake and elimination of florfenicol in Atlantic cod (Gadus morhua) larvae delivered orally through bioencapsulation in the brine shrimp Artemia franciscana
Aquaculture 310 (2010) 27–31
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Uptake and elimination of florfenicol in Atlantic cod (Gadus morhua) larvae delivered orally through bioencapsulation in the brine shrimp Artemia franciscana Irja Sunde Roiha a,⁎, Erling Otterlei b, Amund Litlabø c, Ole Bent Samuelsen a,d a
Institute of Marine Research, P.O. Box 1870 Nordnes, 5817 Bergen, Norway SagaFjord Sea Farm AS, Eldøyane 200, 5411 Stord, Norway Havbruksinstituttet AS, Thormøhlensgate 55, 5008 Bergen, Norway d Section of Pharmacology, Institute of Internal Medicine, University of Bergen, Laboratorieblokken 9 etg. 5021 Bergen, Norway b c
a r t i c l e
i n f o
Article history: Received 12 August 2010 Received in revised form 30 September 2010 Accepted 10 October 2010 Keywords: Antibiotics Enrichment Artemia Live feed Treatment Bacterial disease
a b s t r a c t As an approach to combat bacterial infections, incorporation of florfenicol in Atlantic cod (Gadus morhua) larvae, administered orally through bioencapsulation in Artemia franciscana nauplii, was investigated. Artemia nauplii were enriched with 1000 mg/l florfenicol for 30 min and administered as a single dose, or daily for three consecutive days. The concentration of florfenicol in the medicated nauplii, and in the cod larvae, was analysed by high performance liquid chromatography (HPLC). Following a single oral administration, the elimination half-life (t½β) was calculated to 14 h, Cmax to 4.7 μg/g dry weight (14.3 ng/larva), Tmax to 0.5 h, and MRT to 20 h, respectively. An hour after administration of batch 1 in the triple dose study, a mean florfenicol concentration of 4.6 μg/g dry weight (14.0 ng/larva) was found in the cod larvae. One hour following administration of batch 2 and 3, corresponding values were 5.9 μg/g dry weight (17.9 ng/larva) and 4.2 μg/g dry weight (12.8 ng/larva), respectively. These data show that medication of cod larvae using Artemia nauplii as carriers of florfenicol is a promising approach for treatment. © 2010 Elsevier B.V. All rights reserved.
1. Introduction From 1999 to 2007 the aquaculture production of Atlantic cod (Gadus morhua) in Norway increased from 157 to 11,104 tonnes annually. The majority of the production, 10,375 tons, was based on produced juveniles, whereas only 729 tons were from wild caught fish (Anonymous). In 2007 the number of juveniles produced in hatcheries was 4,867,000 (Norwegian Directorate of Fisheries et al., 2007). The shift from semi-natural rearing systems to intensive rearing of cod has resulted in increased knowledge of larval nutrient requirements, weaning regimes, and the importance of high quality husbandry. However, despite increased knowledge, stable productions of cod larvae are limited by the outbreak of bacterial diseases during the early life stages. As the larvae possess no specific immune system when hatched, due to the immature status of the lymphoid tissues, vaccination is not an option at this stage. Hence, in order to treat an infection, antibacterial therapy is needed. Antibacterial agents may be administered to fish larvae either by bath or orally via the feed. Marine fish larvae are dependent on livefeed organisms at the earliest life stages, and the two most important for use in aquaculture are rotifers (Brachionus plicatilis) and brine ⁎ Corresponding author. Tel.: + 47 55 23 85 57; fax: +47 55 23 85 31. E-mail addresses: [email protected] (I.S. Roiha), [email protected] (E. Otterlei), [email protected] (A. Litlabø), [email protected], [email protected] (O.B. Samuelsen). 0044-8486/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2010.10.014
shrimp (Artemia franciscana) nauplii. Administration of antibacterial agents via rotifers and Artemia nauplii was, therefore, considered an option, and bioencapsulation of a number of antibacterial agents has been investigated. Studied antibacterials for the enrichment of Artemia nauplii are oxytetracycline (OTC) (Touraki et al., 1995; Gomez-Gil et al., 2001; Langdon et al., 2008), erythromycin (Majack et al., 2000; Cook and Rust, 2002; Cook et al., 2003), the combinations of a sulphonamide and either trimethoprim (TMP) or ormethoprim (OMP) (Mohney et al., 1990; Nelis et al., 1991; Verpraet et al., 1992; Chair et al., 1996; Gapasin et al., 1996; Touraki et al., 1996, 1999), and quinolones (Dixon et al., 1995; Touraki et al., 2010). In the majority of these studies, the drugs were administered to Artemia nauplii via liposomes, or lipid emulsions, added to the water, and the enrichment times applied were 4 to 24 h. In hatcheries it is important to start medication as early as possible when an infection is diagnosed. This is due to a rapid increase in the loss of appetite amongst the larvae, and subsequent mortality. Access to a rapid enrichment process is, therefore, of vital importance. The enrichment of Artemia nauplii and rotifers using particles of florfenicol has been investigated by Roiha et al. (2010a, b). They found that the enrichment was fast for both species, and already after 30 min a steady state condition was reached for Artemia nauplii, i.e. no further increase in drug concentration over time was detected (Roiha et al., 2010a). Furthermore, florfenicol possess excellent pharmacokinetic properties in larger fish, including cod (Horsberg et al., 1996; Samuelsen et al., 2003) and in vitro investigations have demonstrated potent activity
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against several bacteria pathogenic to fish (Fukui et al., 1987; Inglis and Richards, 1991). When bath treatment with antibacterial agents was used, the problem with reduced appetite was overcome, but was found to be less effective compared to using enriched Artemia nauplii (Duis et al., 1995; Gapasin et al., 1996; Katharios et al., 2005; Touraki et al., 2010). Studies of the uptake of antibacterial agents in fish larvae, when administered in live feed live-feed, have been performed on European sea bass (Dicentrarchus labrax) and turbot (Scophthalmus maximus) larvae, applying Artemia nauplii enriched with flumequine, oxolinic acid, sarafloxacin, and a combination of trimethoprim (TMP) and sulphamethoxazole (SMX) in the ratio of 1:5 (Chair et al., 1991, 1995; Gapasin et al., 1996; Touraki et al., 1996, 1999, 2010). Whilst the pharmacokinetic properties of florfenicol have been described in larger cod, applying the drug by intravenous injection and orally via medicated feed (Samuelsen et al., 2003; Samuelsen and Bergh, 2004), administration of this antibacterial agent to cod larvae, applying enriched Artemia nauplii, has not been reported. The change from a sole live-feed diet of rotifers to a formulated diet is critical and involves large environmental alterations for the marine larvae. By adding Artemia, as an appetizer, once a day as an afternoon meal, the producer has experienced a better weaning with higher larval survival, and more vigorous and healthy larvae. Also, at an early stage, the size of the rotifers becomes suboptimal as feed for cod larvae, and by a moderate use of Artemia, the larvae are gradually adapted to a diet based exclusively on formulated feed (E. Otterlei, personal communication). The aim of the present study was to investigate the uptake and elimination of florfenicol in cod larvae, when administering the drug via incorporation in nauplii of the live-feed organism A. franciscana. Both single and multiple administrations were examined. 2. Materials and methods 2.1. Chemicals AQUAFLOR®, 50% w/w premix, and the analytical standard of florfenicol were supplied by Intervet/Schering-Plough Animal Health (Summit, NJ, USA). The chloramphenicol standard was purchased from Sigma-Aldrich Norway AS (Oslo, Norway). 1-Heptane sulphonic acid was from Fluka Chemie (Buchs, Switzerland). Methanol (HPLCgrade), acetone, methylene chloride, phosphoric acid (H3PO4), disodium hydrogen phosphate (Na2HPO4), trisodium phosphate (Na3PO4), triethylamine, and n-hexane (all p.a.-grade) were from Merck (Darmstadt, Germany). Standard stock solutions of florfenicol and chloramphenicol were prepared in a concentration of 1 mg/ml in methanol and stored in dark bottles at −20 °C. Working standards were prepared by dilution from the stock solutions with methanol. 2.2. Experimental organisms Cysts of the brine shrimp A. franciscana were purchased from INVE Aquaculture, Inc. (Salt Lake City, Utah, USA). The cysts were hatched in a commercial hatchery, SagaFjord Sea Farm AS (Stord, Norway), using seawater (34–35‰), continuous illumination of 1000 ± 100 lx, and a temperature of 25 ± 1 °C. Twenty-four hours after hatching the nauplii were harvested, separated from the hatching debris, thoroughly rinsed, and made ready for use. The cod larvae were hatched at SagaFjord Sea Farm AS, where the studies were carried out. The larvae were held in circular tanks with a water volume of 7 m3, a temperature of 8.5–10 °C, with constant, weak aeration. Each tank contained approximately 350,000–400,000 larvae. The larvae were reared in an open flow through system, added algae, and were initially fed rotifers and Artemia from day 25 posthatch.
2.3. Enrichment conditions Non-fed Artemia nauplii were enriched with florfenicol using a modified form of AQUAFLOR premix (50%), prepared by adding AQUAFLOR premix to distilled water of approximately 60 °C, in order to dissolve lactose monohydrate and, thereby, liberate the micro-particles of florfenicol. According to the producer, the size of the florfenicol particles will be in the range of 4–10 μm. Since florfenicol has a low solubility in seawater (10 mg/l), the majority of the drug will be present as particles. The enrichments for both experiment 1 and 2 were performed in 60 l plastic tanks; containing seawater with a salinity of 35‰, a temperature of 20 °C, and constant aeration with oxygen levels kept between 70% and 90%. The dosage was 1000 mg/l of active substance, the enrichment time was 30 min, and the density of Artemia nauplii in the enrichment tanks was between 340 and 940 Artemia/ml. Following enrichment, triplicate samples of 500 ml were removed from the container; the nauplii were filtered off using a 150 μm sieve and washed thoroughly with 5 l seawater with a temperature of 20 °C. Two milliliters of the filtrate were stored in an Eppendorf tube at −20 °C until analysed. Using high performance liquid chromatography (HPLC), the various batches of enriched Artemia nauplii were analysed in order to determine the exact concentration of florfenicol per nauplius and, thereby, be able to calculate the average dose given to each cod larva for every administration. 2.4. Pharmacokinetic study At the start of both experiments 1 and 2, the larvae were 35 days old, the average length was about 14 mm, and the weight was approximately 24 mg (wet weight), corresponding to a dry weight of 3 mg (Otterlei et al., 1999; Finn et al., 2002). The cod larvae were fed rotifers (B. plicatilis) three times daily, and once a day a portion of Artemia nauplii. In experiment 1, the cod larvae were fed one portion of 10 million florfenicol-enriched Artemia nauplii at the start of the experiment. This corresponds to an average number of 25 enriched nauplii per cod larva, given that the tanks contain 400,000 larvae. Triplicate samples were collected at 1, 2, 3, 5, 8, 20, and 48 h following initiation of the experiment, and each sample consisted of 100 cod larvae that were transferred to a centrifuge tube and stored at −20 °C until analysed. In experiment 2, the cod larvae were fed a portion of 10 million florfenicol-enriched Artemia nauplii once a day, for 3 consecutive days (batch 1, 2, and 3, respectively). Triplicate samples were collected at 1 and 23 h following administration of batch 1 and 2 and 1 and 48 h following administration of batch 3. One sample consisted of 100 cod larvae stored in a centrifuge tube at −20 °C until analysed. From the relationship between mean drug concentration in cod larvae versus time, in experiment 1, the pharmacokinetic parameters maximum concentration (Cmax), time to maximum concentration (Tmax), elimination half-life (t½) and mean residence time (MRT) were calculated using the program PCNONLIN version 4.2 (Statistical Consultants, Lexington, KY, USA). 2.5. Sampling and sample preparation Artemia nauplii and cod larval samples were prepared and analysed following the procedure described by Roiha et al. (2010a). Prior to sonication in an ultrasonic bath for 10 min, 100 μl of a solution of 1 mg/ml of chloramphenicol in methanol was added to the sample as internal standard. Following sonication, 2.5 ml acetone was added to the homogenate, mixed vigorously by vortexing, and centrifuged at 2500 ×g (5000 rpm) for 10 min, using a Jouan centrifuge (Thermo Scientific, Waltham, MA, USA). The supernatant was extracted with 5 ml methylene chloride, and a 2.2 ml fraction of the extract was evaporated to dryness using nitrogen and a water-bath set at 30 °C.
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The residue was dissolved in 500 μl of a solution of 0.01 M Na2HPO4 (pH 2.8):methanol (80: 20%), washed with 1 ml n-hexane, and filtered through a Spin-X Micro Centrifuge Filter (0.2 μm) from Corning (NY, USA). Twenty microliters (Artemia nauplii) and 50 μl (cod larvae) of the filtrates were used for the HPLC analysis. Artemia nauplii and cod larvae were sampled prior to the initiation of the study and analysed to confirm the absence of florfenicol. Standard curves were prepared for florfenicol in Artemia nauplii in the range of 2 to 100 μg per sample, corresponding to 0.03 to 1.67 ng/nauplius, when counting 61,000 Artemia nauplii per sample, and in cod larvae in the range of 0.01 to 2.5 μg/g, corresponding to 0.24 to 60 ng/larva. Since florfenicol concentrations were calculated for individual organisms in this investigation, the numbers of Artemia nauplii in ten 2 ml samples from each batch were determined prior to the enrichment. The 2 ml sample was added to 2 l seawater and, using aeration, the nauplii were evenly distributed. Five parallels of 200 μl were collected and counted using a magnifying loupe, and the average number of nauplii per 2 ml sample could be calculated. The number of Artemia nauplii in the 2 ml samples varied only to a minor degree when examining 10 parallels and was 61,000 ± 2 000. 2.6. Analytical procedures The HPLC system consisted of an SP 8800 ternary HPLC-pump (Spectra-Physics, San José, CA, USA) connected to a Gilson 234 Autoinjector (Gilson, Middleton, WI, USA) and a Spectra-Physics SP-8480 UV-detector operating at a wavelength of 225 nm. The detector output was coupled to a computerised data system consisting of a Dionex UCI50 Universal Chromatography Interface, the program Dionex Chromeleon Version 6.80 (Dionex Softron, GmbH, Germering, Germany), and a PP04X Dell computer for storage and integration of the chromatograms. The analytical column was a 150 × 4.6 mm Zorbax SB-C-18, 3.5 μm (Agilent Technologies, Karlsruhe, Germany) connected to a short C-18 pre-column (10 × 4.6 mm). The column was operated at room temperature. The mobile phase was a mixture of two solutions, A and B, at ratio 60:40%, respectively. Solution A was made by dissolving 0.02 M heptansulphonate and 0.025 M Na3PO4 in water and adjusting the pH to 3.85 by phosphoric acid, 1 and 5 M. Solution B was methanol containing 0.1% triethylamine. The mobile phase was filtered through a 0.2 μm Millipore filter and degassed using helium and sonication (5 min). The flow rate was 1 ml/min, giving elution times of 5.6 min (florfenicol) and 7.8 min (chloramphenicol). 3. Results The standard curves for florfenicol in Artemia nauplii and cod larvae were linear over the range studied, with r2 = 0. 971 and 0.996 for nauplii and cod larvae, respectively. The limit of detection for the HPLC method was 2 μg/sample for Artemia nauplii and 0.01 μg/g for cod larvae. In the single-dose administration (experiment 1), the concentration of florfenicol in Artemia nauplii was 0.58 ng/nauplius. This corresponds to a dose of 0.7 mg florfenicol per kilogram fish, given that each larva consumes an average of 25 Artemia nauplii. Fig. 1 shows the mean concentrations of florfenicol in cod larvae over time. The highest concentration of 16 ng/larva was found 1 h following feeding, corresponding to concentrations of 0.66 μg/g (wet weight) and 5.3 μg/g (dry weight), using a correlation factor of 8 (Otterlei et al., 1999; Finn et al., 2002). The concentration dropped rapidly, and after 48 h a florfenicol residue of 0.13 μg/g dry weight (0.39 ng/larva) was found. Applying PCNONLIN, the elimination half-life (t½β) was calculated to 14 h, Cmax to 4.7 μg/g dry weight (14.3 ng/larva), Tmax to 0.5 h, and MRT to 20 h, respectively. A number of cod larvae were observed in a loupe to confirm the presence of ingested Artemia nauplii. Thirty minutes following administration, live Artemia nauplii
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Fig. 1. Florfenicol concentration time profile (μg/g dry weight) of 35 day old cod larvae (mean standard length 14 mm and 3 mg dry weight) following a single-dose administration with medicated Artemia nauplii. Each point is a mean ± SD of triplicate samples.
were observed in the stomach of the cod larvae, and after 3 h an orange colour, indicating digested Artemia nauplii, was noticed. For the multiple-dose administration (experiment 2), the concentrations of florfenicol in Artemia nauplii were 1.0, 1.3, and 0.39 ng/ nauplius for batch 1, 2, and 3, respectively, corresponding to daily doses of 1.2, 1.6, and 0.5 mg/kg when an average of 25 nauplii per larva were ingested. An hour after administration of batch 1, a mean florfenicol concentration of 4.6 μg/g dry weight (14.0 ng/larva) was found in the cod larvae. Following administration of batch 2 and 3, corresponding values were 5.9 μg/g dry weight (17.9 ng/larva) and 4.2 μg/g dry weight (12.8 ng/larva), respectively (Fig. 2). 4. Discussion The recommended dosage of florfenicol to treat bacterial infections in larger fish is 10 mg florfenicol per kilogram fish daily, for 10 consecutive days (Nordmo et al., 1998; Lunden et al., 1999; Gaunt, 2004; Samuelsen and Bergh, 2004). At day 35 following hatching, the average length of a cod larva is approximately 14 mm and the wet weight approximately 24 mg/larva (Otterlei et al., 1999; Finn et al., 2002). Hence, in order to achieve the daily dosage, larvae of 24 mg (wet weight) must consume approximately 240 nauplii with a florfenicol concentration of 1.0 ng/nauplius. This is within the recommendations given for cod larvae of that size, since a daily requirement of approximately 300 Artemia nauplii for a 30 day old cod
Fig. 2. Florfenicol concentration time profile (μg/g dry weight) of 35 day old cod larvae following a 3 day administration with medicated Artemia nauplii. The profile shows cod larvae sampled 1 and 23 h after batch 1 and 2, and 1 and 48 h after batch 3. Each point is a mean ± SD of triplicate samples.
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larva increases successively to 3850 when the larva has reached an age of 50 days (Van der Meeren et al., 2005). Similarly, an Atlantic halibut (Hippoglossus hippoglossus) larva with a length of 17 mm requires approximately 500 Artemia nauplii daily, increasing to over 2000 when the larva has reached a length of 22 mm (Gara et al., 1998; Mangor-Jensen, 2004). In this study, the larvae were on a mixed diet of rotifers and Artemia nauplii, and were fed a mean of 25 nauplii/ larva, corresponding to one tenth of a recommended daily dose of drug and number of Artemia nauplii per larva. Except for Cmax, a reduction in dose will, however, not influence the calculation of the pharmacokinetic parameters determined in this study. The highest concentration of florfenicol achieved in Artemia nauplii in this investigation was 1.3 ng/nauplius. In a later experiment, it was shown that when applying a dose of 300 mg/l AQUAFLOR premix blended in seawater and 60 min enrichment time, a florfenicol concentration of 2.49 ng/nauplius was obtained (Roiha et al., 2010a). Thus, the amount of Artemia nauplii needed to achieve a recommended daily dosage can be reduced equivalently. The inter-day variations in the enrichment success obtained in this study were surprising and contradictory to the minor variances obtained in a previous study using the same procedure (Roiha et al., 2010a). We are, therefore, working with a further optimisation of the procedure, examining the importance of age of Artemia nauplii at enrichment, and density of nauplii in the enrichment tanks. This study showed that Artemia nauplii could be used to deliver florfenicol to cod larvae successfully. Peak concentrations of 5.3 μg/g (dry weight) were achieved following a single-dose administration, and 4.6, 5.9, and 4.2 μg/g (dry weight) 1 h following administration of batch 1, 2, and 3, respectively, in the multiple dose study (Figs. 1 and 2). These concentrations are generally lower than the concentrations of antibacterials found in fish larvae in previous conducted studies, and the reason for this is, most probably, the low administered dose in this study. However, since administered dose is rarely given in the earlier studies, a comparison between studies is often difficult to make. Chair et al. (1991) reported on delivery of a combination of TMP and sulphamethoxazole (SMX), in a 1:5 ratio, to 2 month old European sea bass larvae via Artemia nauplii, peak concentrations of 9.69 μg/g (dry weight) of TMP and 12.95 μg/g (dry weight) of SMX 3 h after feeding. In a later study, maximum concentrations of 23.67 and 66.95 μg/g dry weight for TMP and SMX, respectively, were reached 4 h post-feeding in 37 day old turbot larvae (Chair et al., 1995). Touraki et al. (1996) performed a singledose study delivering TMP and SMX to 2 month old sea bass fry, using Artemia nauplii enriched with 64 mg/l of TMP and 256 mg/l of SMX for 24 h before administration to the larvae. Maximum concentrations of drugs in the larvae were 22.71 and 36.21 μg/g wet weight of TMP and SMX, respectively. Duis et al. (1995) investigated the concentrations of oxolinic acid and sarafloxacin in 5 week old turbot larvae, fed medicated Artemia nauplii for ten consecutive days. Peak concentrations in the larvae were 59.1 and 9.1 μg/g dry weight for oxolinic acid and sarafloxacin, respectively. In a recent study, steady state concentration of 80.7 μg/g dry weight of flumequine was achieved after 5 days of oral administration of medicated Artemia nauplii to sea bass larvae (Touraki et al., 2010). An elimination half-life of florfenicol of 14 h in cod larvae is considerably shorter than what is found in larger cod, and suggests that administration of the drug twice a day during medication should be considered. Elimination half-lives of 43 h in plasma, 23 h in liver, and 21 h in muscle were found for florfenicol in cod weighing between 200 and 300 g and held at a temperature of 8 °C (Samuelsen et al., 2003). Other studies have also registered a faster elimination of antibacterials in small compared to larger individuals of the same species. In Atlantic halibut, the elimination half-life of flumequine was calculated to be 10 h in individuals of 3–5 g and 43 h in plasma of larger individuals (1.5– 2.5 kg) (Samuelsen and Lunestad, 1996; Samuelsen and Ervik, 1997). Similarly, the MRT value of 20 h calculated in this study was significantly
lower than the value of 74 h found for larger fish (Samuelsen et al., 2003). The very short Tmax of 0.5 h illustrates that the Artemia nauplii is quickly consumed by the cod larvae. However, at least for the first sampling after 1 h, the florfenicol concentration measured, most probably, represents the amount of drug consumed, and only partly the concentrations in tissues since the observations after 30 min showed undigested Artemia nauplii in the stomach of the cod larvae. In larger cod, plasma Tmax of 7 h was found, following oral administration of medicated feed, showing that florfenicol is relatively fast absorbed in larger cod (Samuelsen et al., 2003). In the same investigation, Tmax values of 9 h were found, in both muscle and liver of the fish, showing a fast distribution from plasma to tissues (Samuelsen et al., 2003). The results from the multiple-administration (Fig. 2) verify the results from the single administration. The decrease in concentration, seen in the larvae from day 2 to 3, is caused by the lower concentration achieved in batch 3, following enrichment of Artemia nauplii. In conclusion, the data show that medication with florfenicol, using Artemia nauplii as carriers and an enrichment time of 30 min, is a promising approach for the treatment of bacterial infections in cod larvae. Acknowledgment This work was funded by the Norwegian Research Council project no. 168463, PROPHYLHATCH. The technical assistance of Audun Høylandsskjær and Ina Nepstad is highly appreciated. References Chair, M., Romdhane, M., Dehasque, M., Nelis, H., De Leenheer, A.P., Sorgeloos, P., 1991. Live-food mediated drug delivery as a tool for disease treatment in larviculture. II. A case study with European seabass. In: Lavens, P., Sorgeloos, P., Jaspers, E., Ollevier, F. (Eds.), Larvi '91—Symposium on Fish and Crustacean Larviculture. European Aquaculture Society Special Publication Gent, Belgium, pp. 412–414. Chair, M., Dehasque, M., Sorgeloos, P., Nelis, H., De Leenheer, A.P., 1995. Live food mediated drug delivery as a tool for disease treatment in larviculture: a case study with turbot Scophthalmus maximus. Journal of the World Aquaculture Society 26, 217–219. Chair, M., Nelis, H.J., Leger, P., Sorgeloos, P., De Leenheer, A.P., 1996. Accumulation of trimethoprim, sulfamethoxazole, and N-acetylsulfamethoxazole in fish and shrimp fed medicated Artemia franciscana. Antimicrobial Agents and Chemotherapy 40, 1649–1652. Cook, M.A., Rust, M.B., 2002. Bioencapsulation of five forms of erythromycin by adult Artemia salina (L.). Journal of Fish Diseases 25, 165–170. Cook, M.A., Rust, M.B., Massee, K., Majack, T., Peterson, M.E., 2003. Uptake of erythromycin by first-feeding sockeye salmon, Oncorhynchus nerka (Walbaum), fed live or freeze-dried enriched adult Artemia or medicated pellets. Journal of Fish Diseases 26, 277–285. Dixon, B.A., Van Poucke, S.O., Chair, M., Dehasque, M., Nelis, H.J., Sorgeloos, P., De Leenheer, A.P., 1995. COMMUNICATIONS: Bioencapsulation of the antibacterial drug sarafloxacin in nauplii of the brine shrimp Artemia franciscana. Journal of Aquatic Animal Health 7, 42–45. Duis, K., Hammer, C., Beveridge, M.C.M., Inglis, V., Braum, E., 1995. Delivery of quinolone antibacterials to turbot, Scophthalmus maximus (L.), via bioencapsulation: quantification and efficacy trial. Journal of Fish Diseases 18, 229–238. Finn, R.N., Rønnestad, I., van der Meeren, T., Fyhn, H.J., 2002. Fuel and metabolic scaling during the early life stages of Atlantic cod Gadus morhua. Marine Ecology-Progress Series 243, 217–234. Fukui, H., Fujihara, Y., Kano, T., 1987. In vitro and In vivo antibacterial activities of florfenicol, a new fluorinated analog of thiamphenicol, against fish pathogens. Fish Pathology 22, 201–207. Gapasin, R.S.J., Nelis, H.J., Chair, M., Sorgeloos, P., 1996. Drug assimilation in the tissue of European sea bass (Dicentrarchus labrax) fry delivered orally through bioencapsulation. Journal of Applied Ichthyology (Zeitschrift Fur Angewandte Ichthyologie) 12, 39–42. Gara, B., Shields, R.J., McEvoy, L., 1998. Feeding strategies to achieve correct metamorphosis of Atlantic halibut, Hippoglossus hippoglossus L., using enriched Artemia. Aquaculture Research 29, 935–948. Gaunt, P.S., 2004. Determination of dose rate of florfenicol in feed for control of mortality in channel catfish Ictalurus punctatus (Rafinesque) infected with Edwardsiella ictaluri, etiological agent of enteric septicemia. Journal of the World Aquaculture Society 35, 257–267. Gomez-Gil, B., Cabanillas-Ramos, J., Paez-Brambila, S., Roque, A., 2001. Standardization of the bioencapsulation of enrofloxacin and oxytetracycline in Artemia franciscana Kellogg, 1906. Aquaculture 196, 1–12.
I.S. Roiha et al. / Aquaculture 310 (2010) 27–31 Horsberg, T.E., Hoff, K.A., Nordmo, R., 1996. Pharmacokinetics of florfenicol and its metabolite florfenicol amine in Atlantic salmon. Journal of Aquatic Animal Health 8, 292–301. Inglis, V., Richards, R.H., 1991. The in vitro susceptibility of Aeromonas salmonicida and other fish-pathogenic bacteria to 29 antimicrobial agents. Journal of Fish Diseases 14, 641–650. Katharios, P., Smullen, R.P., Inglis, V., 2005. The use of the polychaete worm Nereis virens eggs as vehicle for the delivery of oxytetracycline in Solea solea larvae. Aquaculture 243, 1–7. Langdon, C., Nordgreen, A., Hawkyard, M., Hamre, K., 2008. Evaluation of wax spray beads for delivery of low-molecular weight, water-soluble nutrients and antibiotics to Artemia. Aquaculture 284, 151–158. Lunden, T., Miettinen, S., Lonnstrom, L.G., Lilius, E.M., Bylund, C., 1999. Effect of florfenicol on the immune response of rainbow trout (Oncorhynchus mykiss). Veterinary Immunology and Immunopathology 67, 317–325. Majack, T.J., Rust, M.B., Massee, K.C., Kissil, G.W., Hardy, R.W., Peterson, M.E., 2000. Bioencapsulation of erythromycin using adult brine shrimp, Artemia franciscana (Latreille). Journal of Fish Diseases 23, 71–76. Mangor-Jensen, A., 2004. Første fødeopptak. In: Holm, J.C., Mangor-Jensen, A. (Eds.), Håndbok i kveiteoppdrett. Institute of Marine Research, Bergen, pp. 45–55. Mohney, L.L., Lightner, D.V.L., Williams, R.R., Bauerlein, M., 1990. Bioencapsulation of therapeutic quantities of the antibacterial Romet-30 in nauplii of the brine shrimp Artemia and in the nematode Panagrellus redivivus. Journal of the World Aquaculture Society 21, 186–191. Nelis, H.J., Leger, F., Sorgeloos, P., Deleenheer, A.P., 1991. Liquid-chromatographic determination of efficacy of incorporation of trimethoprim and sulfamethoxazole in brine shrimp (Artemia spp) used for prophylactic chemotherapy of fish. Antimicrobial Agents and Chemotherapy 35, 2486–2489. Nordmo, R., Riseth, J.M.H., Varma, K.J., Sutherland, I.H., Brokken, E.S., 1998. Evaluation of florfenicol in Atlantic salmon, Salmo salar L: efficacy against furunculosis due to Aeromonas salmonicida and cold water vibriosis due to Vibrio salmonicida. Journal of Fish Diseases 21, 289–297. Norwegian Directorate of Fisheries, Aquaculture Department, Statistics Department, 2007. Nøkkeltall fra norsk havbruksnæring. Norwegian Directorate of Fisheries, Bergen, Norway. Otterlei, E., Nyhammer, G., Folkvord, A., Stefansson, S.O., 1999. Temperature- and sizedependent growth of larval and early juvenile Atlantic cod (Gadus morhua): a comparative study of Norwegian coastal cod and northeast Arctic cod. Canadian Journal of Fisheries and Aquatic Sciences 56, 2099–2111.
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Roiha, I.S., Otterlei, E., Samuelsen, O., 2010a. Bioencapsulation of florfenicol in brine shrimp, Artemia franciscana, nauplii. Journal of Bioanalysis and Biomedicine 2, 060–064. Roiha, I.S., Otterlei, E., Samuelsen, O.B., 2010b. Evaluating bioencapsulation of florfenicol in rotifers (Brachionus plicatilis). Aquaculture Research. doi:10.1111/ j.1365-2109.2010.02697.x. Samuelsen, O.B., Bergh, O., 2004. Efficacy of orally administered florfenicol and oxolinic acid for the treatment of vibriosis in cod (Gadus morhua). Aquaculture 235, 27–35. Samuelsen, O.B., Ervik, A., 1997. Single dose pharmacokinetic study of flumequine after intravenous, intraperitoneal and oral administration to Atlantic halibut (Hippoglossus hippoglossus) held in seawater at 9 degrees C. Aquaculture 158, 215–227. Samuelsen, O.B., Lunestad, B.T., 1996. Bath treatment, an alternative method for the administration of the quinolones flumequine and oxolinic acid to halibut Hippoglossus hippoglossus, and in vitro antibacterial activity of the drugs against some Vibrio sp. Diseases of Aquatic Organisms 27, 13–18. Samuelsen, O.B., Bergh, O., Ervik, A., 2003. Pharmacokinetics of florfenicol in cod Gadus morhua and in vitro antibacterial activity against Vibrio anguillarum. Diseases of Aquatic Organisms 56, 127–133. Touraki, M., Rigas, P., Kastritsis, C., 1995. Liposome mediated delivery of water soluble antibiotics to the larvae of aquatic animals. Aquaculture 136, 1–10. Touraki, M., Mourelatos, S., Karamanlidou, G., Kalaitzopoulou, S., Kastritsis, C., 1996. Bioencapsulation of chemotherapeutics in Artemia as a means of prevention and treatment of infectious diseases of marine fish fry. Aquacultural Engineering 15, 133–147. Touraki, M., Niopas, I., Kastritsis, C., 1999. Bioaccumulation of trimethoprim, sulfamethoxazole and N-acetyl-sulfamethoxazole in Artemia nauplii and residual kinetics in seabass larvae after repeated oral dosing of medicated nauplii. Aquaculture 175, 15–30. Touraki, M., Niopas, I., Ladoukakis, E., Karagiannis, V., 2010. Efficacy of flumequine administered by bath or through medicated nauplii of Artemia fransiscana (L.) in the treatment of vibriosis in sea bass larvae. Aquaculture 306, 146–152. Van der Meeren, T., Pedersen, J.P., Kolbeinshavn, A.G., 2005. Yngelproduksjon— larvefase. In: Otterå, H., Borthen, J., Taranger, G.L. (Eds.), Oppdrett av torsk: næring med framtid. Norsk fiskeoppdrett AS, Bergen, pp. 85–111. Verpraet, R., Chair, M., Leger, P., Nelis, H., Sorgeloos, P., Deleenheer, A., 1992. Live-food mediated drug delivery as a tool for disease treatment in larviculture. The enrichment of therapeutics in rotifers and Artemia nauplii. Aquacultural Engineering 11, 133–139.
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Aquaculture j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / a q u a - o n l i n e
Uptake and elimination of florfenicol in Atlantic cod (Gadus morhua) larvae delivered orally through bioencapsulation in the brine shrimp Artemia franciscana Irja Sunde Roiha a,⁎, Erling Otterlei b, Amund Litlabø c, Ole Bent Samuelsen a,d a
Institute of Marine Research, P.O. Box 1870 Nordnes, 5817 Bergen, Norway SagaFjord Sea Farm AS, Eldøyane 200, 5411 Stord, Norway Havbruksinstituttet AS, Thormøhlensgate 55, 5008 Bergen, Norway d Section of Pharmacology, Institute of Internal Medicine, University of Bergen, Laboratorieblokken 9 etg. 5021 Bergen, Norway b c
a r t i c l e
i n f o
Article history: Received 12 August 2010 Received in revised form 30 September 2010 Accepted 10 October 2010 Keywords: Antibiotics Enrichment Artemia Live feed Treatment Bacterial disease
a b s t r a c t As an approach to combat bacterial infections, incorporation of florfenicol in Atlantic cod (Gadus morhua) larvae, administered orally through bioencapsulation in Artemia franciscana nauplii, was investigated. Artemia nauplii were enriched with 1000 mg/l florfenicol for 30 min and administered as a single dose, or daily for three consecutive days. The concentration of florfenicol in the medicated nauplii, and in the cod larvae, was analysed by high performance liquid chromatography (HPLC). Following a single oral administration, the elimination half-life (t½β) was calculated to 14 h, Cmax to 4.7 μg/g dry weight (14.3 ng/larva), Tmax to 0.5 h, and MRT to 20 h, respectively. An hour after administration of batch 1 in the triple dose study, a mean florfenicol concentration of 4.6 μg/g dry weight (14.0 ng/larva) was found in the cod larvae. One hour following administration of batch 2 and 3, corresponding values were 5.9 μg/g dry weight (17.9 ng/larva) and 4.2 μg/g dry weight (12.8 ng/larva), respectively. These data show that medication of cod larvae using Artemia nauplii as carriers of florfenicol is a promising approach for treatment. © 2010 Elsevier B.V. All rights reserved.
1. Introduction From 1999 to 2007 the aquaculture production of Atlantic cod (Gadus morhua) in Norway increased from 157 to 11,104 tonnes annually. The majority of the production, 10,375 tons, was based on produced juveniles, whereas only 729 tons were from wild caught fish (Anonymous). In 2007 the number of juveniles produced in hatcheries was 4,867,000 (Norwegian Directorate of Fisheries et al., 2007). The shift from semi-natural rearing systems to intensive rearing of cod has resulted in increased knowledge of larval nutrient requirements, weaning regimes, and the importance of high quality husbandry. However, despite increased knowledge, stable productions of cod larvae are limited by the outbreak of bacterial diseases during the early life stages. As the larvae possess no specific immune system when hatched, due to the immature status of the lymphoid tissues, vaccination is not an option at this stage. Hence, in order to treat an infection, antibacterial therapy is needed. Antibacterial agents may be administered to fish larvae either by bath or orally via the feed. Marine fish larvae are dependent on livefeed organisms at the earliest life stages, and the two most important for use in aquaculture are rotifers (Brachionus plicatilis) and brine ⁎ Corresponding author. Tel.: + 47 55 23 85 57; fax: +47 55 23 85 31. E-mail addresses: [email protected] (I.S. Roiha), [email protected] (E. Otterlei), [email protected] (A. Litlabø), [email protected], [email protected] (O.B. Samuelsen). 0044-8486/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2010.10.014
shrimp (Artemia franciscana) nauplii. Administration of antibacterial agents via rotifers and Artemia nauplii was, therefore, considered an option, and bioencapsulation of a number of antibacterial agents has been investigated. Studied antibacterials for the enrichment of Artemia nauplii are oxytetracycline (OTC) (Touraki et al., 1995; Gomez-Gil et al., 2001; Langdon et al., 2008), erythromycin (Majack et al., 2000; Cook and Rust, 2002; Cook et al., 2003), the combinations of a sulphonamide and either trimethoprim (TMP) or ormethoprim (OMP) (Mohney et al., 1990; Nelis et al., 1991; Verpraet et al., 1992; Chair et al., 1996; Gapasin et al., 1996; Touraki et al., 1996, 1999), and quinolones (Dixon et al., 1995; Touraki et al., 2010). In the majority of these studies, the drugs were administered to Artemia nauplii via liposomes, or lipid emulsions, added to the water, and the enrichment times applied were 4 to 24 h. In hatcheries it is important to start medication as early as possible when an infection is diagnosed. This is due to a rapid increase in the loss of appetite amongst the larvae, and subsequent mortality. Access to a rapid enrichment process is, therefore, of vital importance. The enrichment of Artemia nauplii and rotifers using particles of florfenicol has been investigated by Roiha et al. (2010a, b). They found that the enrichment was fast for both species, and already after 30 min a steady state condition was reached for Artemia nauplii, i.e. no further increase in drug concentration over time was detected (Roiha et al., 2010a). Furthermore, florfenicol possess excellent pharmacokinetic properties in larger fish, including cod (Horsberg et al., 1996; Samuelsen et al., 2003) and in vitro investigations have demonstrated potent activity
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against several bacteria pathogenic to fish (Fukui et al., 1987; Inglis and Richards, 1991). When bath treatment with antibacterial agents was used, the problem with reduced appetite was overcome, but was found to be less effective compared to using enriched Artemia nauplii (Duis et al., 1995; Gapasin et al., 1996; Katharios et al., 2005; Touraki et al., 2010). Studies of the uptake of antibacterial agents in fish larvae, when administered in live feed live-feed, have been performed on European sea bass (Dicentrarchus labrax) and turbot (Scophthalmus maximus) larvae, applying Artemia nauplii enriched with flumequine, oxolinic acid, sarafloxacin, and a combination of trimethoprim (TMP) and sulphamethoxazole (SMX) in the ratio of 1:5 (Chair et al., 1991, 1995; Gapasin et al., 1996; Touraki et al., 1996, 1999, 2010). Whilst the pharmacokinetic properties of florfenicol have been described in larger cod, applying the drug by intravenous injection and orally via medicated feed (Samuelsen et al., 2003; Samuelsen and Bergh, 2004), administration of this antibacterial agent to cod larvae, applying enriched Artemia nauplii, has not been reported. The change from a sole live-feed diet of rotifers to a formulated diet is critical and involves large environmental alterations for the marine larvae. By adding Artemia, as an appetizer, once a day as an afternoon meal, the producer has experienced a better weaning with higher larval survival, and more vigorous and healthy larvae. Also, at an early stage, the size of the rotifers becomes suboptimal as feed for cod larvae, and by a moderate use of Artemia, the larvae are gradually adapted to a diet based exclusively on formulated feed (E. Otterlei, personal communication). The aim of the present study was to investigate the uptake and elimination of florfenicol in cod larvae, when administering the drug via incorporation in nauplii of the live-feed organism A. franciscana. Both single and multiple administrations were examined. 2. Materials and methods 2.1. Chemicals AQUAFLOR®, 50% w/w premix, and the analytical standard of florfenicol were supplied by Intervet/Schering-Plough Animal Health (Summit, NJ, USA). The chloramphenicol standard was purchased from Sigma-Aldrich Norway AS (Oslo, Norway). 1-Heptane sulphonic acid was from Fluka Chemie (Buchs, Switzerland). Methanol (HPLCgrade), acetone, methylene chloride, phosphoric acid (H3PO4), disodium hydrogen phosphate (Na2HPO4), trisodium phosphate (Na3PO4), triethylamine, and n-hexane (all p.a.-grade) were from Merck (Darmstadt, Germany). Standard stock solutions of florfenicol and chloramphenicol were prepared in a concentration of 1 mg/ml in methanol and stored in dark bottles at −20 °C. Working standards were prepared by dilution from the stock solutions with methanol. 2.2. Experimental organisms Cysts of the brine shrimp A. franciscana were purchased from INVE Aquaculture, Inc. (Salt Lake City, Utah, USA). The cysts were hatched in a commercial hatchery, SagaFjord Sea Farm AS (Stord, Norway), using seawater (34–35‰), continuous illumination of 1000 ± 100 lx, and a temperature of 25 ± 1 °C. Twenty-four hours after hatching the nauplii were harvested, separated from the hatching debris, thoroughly rinsed, and made ready for use. The cod larvae were hatched at SagaFjord Sea Farm AS, where the studies were carried out. The larvae were held in circular tanks with a water volume of 7 m3, a temperature of 8.5–10 °C, with constant, weak aeration. Each tank contained approximately 350,000–400,000 larvae. The larvae were reared in an open flow through system, added algae, and were initially fed rotifers and Artemia from day 25 posthatch.
2.3. Enrichment conditions Non-fed Artemia nauplii were enriched with florfenicol using a modified form of AQUAFLOR premix (50%), prepared by adding AQUAFLOR premix to distilled water of approximately 60 °C, in order to dissolve lactose monohydrate and, thereby, liberate the micro-particles of florfenicol. According to the producer, the size of the florfenicol particles will be in the range of 4–10 μm. Since florfenicol has a low solubility in seawater (10 mg/l), the majority of the drug will be present as particles. The enrichments for both experiment 1 and 2 were performed in 60 l plastic tanks; containing seawater with a salinity of 35‰, a temperature of 20 °C, and constant aeration with oxygen levels kept between 70% and 90%. The dosage was 1000 mg/l of active substance, the enrichment time was 30 min, and the density of Artemia nauplii in the enrichment tanks was between 340 and 940 Artemia/ml. Following enrichment, triplicate samples of 500 ml were removed from the container; the nauplii were filtered off using a 150 μm sieve and washed thoroughly with 5 l seawater with a temperature of 20 °C. Two milliliters of the filtrate were stored in an Eppendorf tube at −20 °C until analysed. Using high performance liquid chromatography (HPLC), the various batches of enriched Artemia nauplii were analysed in order to determine the exact concentration of florfenicol per nauplius and, thereby, be able to calculate the average dose given to each cod larva for every administration. 2.4. Pharmacokinetic study At the start of both experiments 1 and 2, the larvae were 35 days old, the average length was about 14 mm, and the weight was approximately 24 mg (wet weight), corresponding to a dry weight of 3 mg (Otterlei et al., 1999; Finn et al., 2002). The cod larvae were fed rotifers (B. plicatilis) three times daily, and once a day a portion of Artemia nauplii. In experiment 1, the cod larvae were fed one portion of 10 million florfenicol-enriched Artemia nauplii at the start of the experiment. This corresponds to an average number of 25 enriched nauplii per cod larva, given that the tanks contain 400,000 larvae. Triplicate samples were collected at 1, 2, 3, 5, 8, 20, and 48 h following initiation of the experiment, and each sample consisted of 100 cod larvae that were transferred to a centrifuge tube and stored at −20 °C until analysed. In experiment 2, the cod larvae were fed a portion of 10 million florfenicol-enriched Artemia nauplii once a day, for 3 consecutive days (batch 1, 2, and 3, respectively). Triplicate samples were collected at 1 and 23 h following administration of batch 1 and 2 and 1 and 48 h following administration of batch 3. One sample consisted of 100 cod larvae stored in a centrifuge tube at −20 °C until analysed. From the relationship between mean drug concentration in cod larvae versus time, in experiment 1, the pharmacokinetic parameters maximum concentration (Cmax), time to maximum concentration (Tmax), elimination half-life (t½) and mean residence time (MRT) were calculated using the program PCNONLIN version 4.2 (Statistical Consultants, Lexington, KY, USA). 2.5. Sampling and sample preparation Artemia nauplii and cod larval samples were prepared and analysed following the procedure described by Roiha et al. (2010a). Prior to sonication in an ultrasonic bath for 10 min, 100 μl of a solution of 1 mg/ml of chloramphenicol in methanol was added to the sample as internal standard. Following sonication, 2.5 ml acetone was added to the homogenate, mixed vigorously by vortexing, and centrifuged at 2500 ×g (5000 rpm) for 10 min, using a Jouan centrifuge (Thermo Scientific, Waltham, MA, USA). The supernatant was extracted with 5 ml methylene chloride, and a 2.2 ml fraction of the extract was evaporated to dryness using nitrogen and a water-bath set at 30 °C.
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The residue was dissolved in 500 μl of a solution of 0.01 M Na2HPO4 (pH 2.8):methanol (80: 20%), washed with 1 ml n-hexane, and filtered through a Spin-X Micro Centrifuge Filter (0.2 μm) from Corning (NY, USA). Twenty microliters (Artemia nauplii) and 50 μl (cod larvae) of the filtrates were used for the HPLC analysis. Artemia nauplii and cod larvae were sampled prior to the initiation of the study and analysed to confirm the absence of florfenicol. Standard curves were prepared for florfenicol in Artemia nauplii in the range of 2 to 100 μg per sample, corresponding to 0.03 to 1.67 ng/nauplius, when counting 61,000 Artemia nauplii per sample, and in cod larvae in the range of 0.01 to 2.5 μg/g, corresponding to 0.24 to 60 ng/larva. Since florfenicol concentrations were calculated for individual organisms in this investigation, the numbers of Artemia nauplii in ten 2 ml samples from each batch were determined prior to the enrichment. The 2 ml sample was added to 2 l seawater and, using aeration, the nauplii were evenly distributed. Five parallels of 200 μl were collected and counted using a magnifying loupe, and the average number of nauplii per 2 ml sample could be calculated. The number of Artemia nauplii in the 2 ml samples varied only to a minor degree when examining 10 parallels and was 61,000 ± 2 000. 2.6. Analytical procedures The HPLC system consisted of an SP 8800 ternary HPLC-pump (Spectra-Physics, San José, CA, USA) connected to a Gilson 234 Autoinjector (Gilson, Middleton, WI, USA) and a Spectra-Physics SP-8480 UV-detector operating at a wavelength of 225 nm. The detector output was coupled to a computerised data system consisting of a Dionex UCI50 Universal Chromatography Interface, the program Dionex Chromeleon Version 6.80 (Dionex Softron, GmbH, Germering, Germany), and a PP04X Dell computer for storage and integration of the chromatograms. The analytical column was a 150 × 4.6 mm Zorbax SB-C-18, 3.5 μm (Agilent Technologies, Karlsruhe, Germany) connected to a short C-18 pre-column (10 × 4.6 mm). The column was operated at room temperature. The mobile phase was a mixture of two solutions, A and B, at ratio 60:40%, respectively. Solution A was made by dissolving 0.02 M heptansulphonate and 0.025 M Na3PO4 in water and adjusting the pH to 3.85 by phosphoric acid, 1 and 5 M. Solution B was methanol containing 0.1% triethylamine. The mobile phase was filtered through a 0.2 μm Millipore filter and degassed using helium and sonication (5 min). The flow rate was 1 ml/min, giving elution times of 5.6 min (florfenicol) and 7.8 min (chloramphenicol). 3. Results The standard curves for florfenicol in Artemia nauplii and cod larvae were linear over the range studied, with r2 = 0. 971 and 0.996 for nauplii and cod larvae, respectively. The limit of detection for the HPLC method was 2 μg/sample for Artemia nauplii and 0.01 μg/g for cod larvae. In the single-dose administration (experiment 1), the concentration of florfenicol in Artemia nauplii was 0.58 ng/nauplius. This corresponds to a dose of 0.7 mg florfenicol per kilogram fish, given that each larva consumes an average of 25 Artemia nauplii. Fig. 1 shows the mean concentrations of florfenicol in cod larvae over time. The highest concentration of 16 ng/larva was found 1 h following feeding, corresponding to concentrations of 0.66 μg/g (wet weight) and 5.3 μg/g (dry weight), using a correlation factor of 8 (Otterlei et al., 1999; Finn et al., 2002). The concentration dropped rapidly, and after 48 h a florfenicol residue of 0.13 μg/g dry weight (0.39 ng/larva) was found. Applying PCNONLIN, the elimination half-life (t½β) was calculated to 14 h, Cmax to 4.7 μg/g dry weight (14.3 ng/larva), Tmax to 0.5 h, and MRT to 20 h, respectively. A number of cod larvae were observed in a loupe to confirm the presence of ingested Artemia nauplii. Thirty minutes following administration, live Artemia nauplii
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Fig. 1. Florfenicol concentration time profile (μg/g dry weight) of 35 day old cod larvae (mean standard length 14 mm and 3 mg dry weight) following a single-dose administration with medicated Artemia nauplii. Each point is a mean ± SD of triplicate samples.
were observed in the stomach of the cod larvae, and after 3 h an orange colour, indicating digested Artemia nauplii, was noticed. For the multiple-dose administration (experiment 2), the concentrations of florfenicol in Artemia nauplii were 1.0, 1.3, and 0.39 ng/ nauplius for batch 1, 2, and 3, respectively, corresponding to daily doses of 1.2, 1.6, and 0.5 mg/kg when an average of 25 nauplii per larva were ingested. An hour after administration of batch 1, a mean florfenicol concentration of 4.6 μg/g dry weight (14.0 ng/larva) was found in the cod larvae. Following administration of batch 2 and 3, corresponding values were 5.9 μg/g dry weight (17.9 ng/larva) and 4.2 μg/g dry weight (12.8 ng/larva), respectively (Fig. 2). 4. Discussion The recommended dosage of florfenicol to treat bacterial infections in larger fish is 10 mg florfenicol per kilogram fish daily, for 10 consecutive days (Nordmo et al., 1998; Lunden et al., 1999; Gaunt, 2004; Samuelsen and Bergh, 2004). At day 35 following hatching, the average length of a cod larva is approximately 14 mm and the wet weight approximately 24 mg/larva (Otterlei et al., 1999; Finn et al., 2002). Hence, in order to achieve the daily dosage, larvae of 24 mg (wet weight) must consume approximately 240 nauplii with a florfenicol concentration of 1.0 ng/nauplius. This is within the recommendations given for cod larvae of that size, since a daily requirement of approximately 300 Artemia nauplii for a 30 day old cod
Fig. 2. Florfenicol concentration time profile (μg/g dry weight) of 35 day old cod larvae following a 3 day administration with medicated Artemia nauplii. The profile shows cod larvae sampled 1 and 23 h after batch 1 and 2, and 1 and 48 h after batch 3. Each point is a mean ± SD of triplicate samples.
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larva increases successively to 3850 when the larva has reached an age of 50 days (Van der Meeren et al., 2005). Similarly, an Atlantic halibut (Hippoglossus hippoglossus) larva with a length of 17 mm requires approximately 500 Artemia nauplii daily, increasing to over 2000 when the larva has reached a length of 22 mm (Gara et al., 1998; Mangor-Jensen, 2004). In this study, the larvae were on a mixed diet of rotifers and Artemia nauplii, and were fed a mean of 25 nauplii/ larva, corresponding to one tenth of a recommended daily dose of drug and number of Artemia nauplii per larva. Except for Cmax, a reduction in dose will, however, not influence the calculation of the pharmacokinetic parameters determined in this study. The highest concentration of florfenicol achieved in Artemia nauplii in this investigation was 1.3 ng/nauplius. In a later experiment, it was shown that when applying a dose of 300 mg/l AQUAFLOR premix blended in seawater and 60 min enrichment time, a florfenicol concentration of 2.49 ng/nauplius was obtained (Roiha et al., 2010a). Thus, the amount of Artemia nauplii needed to achieve a recommended daily dosage can be reduced equivalently. The inter-day variations in the enrichment success obtained in this study were surprising and contradictory to the minor variances obtained in a previous study using the same procedure (Roiha et al., 2010a). We are, therefore, working with a further optimisation of the procedure, examining the importance of age of Artemia nauplii at enrichment, and density of nauplii in the enrichment tanks. This study showed that Artemia nauplii could be used to deliver florfenicol to cod larvae successfully. Peak concentrations of 5.3 μg/g (dry weight) were achieved following a single-dose administration, and 4.6, 5.9, and 4.2 μg/g (dry weight) 1 h following administration of batch 1, 2, and 3, respectively, in the multiple dose study (Figs. 1 and 2). These concentrations are generally lower than the concentrations of antibacterials found in fish larvae in previous conducted studies, and the reason for this is, most probably, the low administered dose in this study. However, since administered dose is rarely given in the earlier studies, a comparison between studies is often difficult to make. Chair et al. (1991) reported on delivery of a combination of TMP and sulphamethoxazole (SMX), in a 1:5 ratio, to 2 month old European sea bass larvae via Artemia nauplii, peak concentrations of 9.69 μg/g (dry weight) of TMP and 12.95 μg/g (dry weight) of SMX 3 h after feeding. In a later study, maximum concentrations of 23.67 and 66.95 μg/g dry weight for TMP and SMX, respectively, were reached 4 h post-feeding in 37 day old turbot larvae (Chair et al., 1995). Touraki et al. (1996) performed a singledose study delivering TMP and SMX to 2 month old sea bass fry, using Artemia nauplii enriched with 64 mg/l of TMP and 256 mg/l of SMX for 24 h before administration to the larvae. Maximum concentrations of drugs in the larvae were 22.71 and 36.21 μg/g wet weight of TMP and SMX, respectively. Duis et al. (1995) investigated the concentrations of oxolinic acid and sarafloxacin in 5 week old turbot larvae, fed medicated Artemia nauplii for ten consecutive days. Peak concentrations in the larvae were 59.1 and 9.1 μg/g dry weight for oxolinic acid and sarafloxacin, respectively. In a recent study, steady state concentration of 80.7 μg/g dry weight of flumequine was achieved after 5 days of oral administration of medicated Artemia nauplii to sea bass larvae (Touraki et al., 2010). An elimination half-life of florfenicol of 14 h in cod larvae is considerably shorter than what is found in larger cod, and suggests that administration of the drug twice a day during medication should be considered. Elimination half-lives of 43 h in plasma, 23 h in liver, and 21 h in muscle were found for florfenicol in cod weighing between 200 and 300 g and held at a temperature of 8 °C (Samuelsen et al., 2003). Other studies have also registered a faster elimination of antibacterials in small compared to larger individuals of the same species. In Atlantic halibut, the elimination half-life of flumequine was calculated to be 10 h in individuals of 3–5 g and 43 h in plasma of larger individuals (1.5– 2.5 kg) (Samuelsen and Lunestad, 1996; Samuelsen and Ervik, 1997). Similarly, the MRT value of 20 h calculated in this study was significantly
lower than the value of 74 h found for larger fish (Samuelsen et al., 2003). The very short Tmax of 0.5 h illustrates that the Artemia nauplii is quickly consumed by the cod larvae. However, at least for the first sampling after 1 h, the florfenicol concentration measured, most probably, represents the amount of drug consumed, and only partly the concentrations in tissues since the observations after 30 min showed undigested Artemia nauplii in the stomach of the cod larvae. In larger cod, plasma Tmax of 7 h was found, following oral administration of medicated feed, showing that florfenicol is relatively fast absorbed in larger cod (Samuelsen et al., 2003). In the same investigation, Tmax values of 9 h were found, in both muscle and liver of the fish, showing a fast distribution from plasma to tissues (Samuelsen et al., 2003). The results from the multiple-administration (Fig. 2) verify the results from the single administration. The decrease in concentration, seen in the larvae from day 2 to 3, is caused by the lower concentration achieved in batch 3, following enrichment of Artemia nauplii. In conclusion, the data show that medication with florfenicol, using Artemia nauplii as carriers and an enrichment time of 30 min, is a promising approach for the treatment of bacterial infections in cod larvae. Acknowledgment This work was funded by the Norwegian Research Council project no. 168463, PROPHYLHATCH. The technical assistance of Audun Høylandsskjær and Ina Nepstad is highly appreciated. References Chair, M., Romdhane, M., Dehasque, M., Nelis, H., De Leenheer, A.P., Sorgeloos, P., 1991. Live-food mediated drug delivery as a tool for disease treatment in larviculture. II. A case study with European seabass. In: Lavens, P., Sorgeloos, P., Jaspers, E., Ollevier, F. (Eds.), Larvi '91—Symposium on Fish and Crustacean Larviculture. European Aquaculture Society Special Publication Gent, Belgium, pp. 412–414. Chair, M., Dehasque, M., Sorgeloos, P., Nelis, H., De Leenheer, A.P., 1995. Live food mediated drug delivery as a tool for disease treatment in larviculture: a case study with turbot Scophthalmus maximus. Journal of the World Aquaculture Society 26, 217–219. Chair, M., Nelis, H.J., Leger, P., Sorgeloos, P., De Leenheer, A.P., 1996. Accumulation of trimethoprim, sulfamethoxazole, and N-acetylsulfamethoxazole in fish and shrimp fed medicated Artemia franciscana. Antimicrobial Agents and Chemotherapy 40, 1649–1652. Cook, M.A., Rust, M.B., 2002. Bioencapsulation of five forms of erythromycin by adult Artemia salina (L.). Journal of Fish Diseases 25, 165–170. Cook, M.A., Rust, M.B., Massee, K., Majack, T., Peterson, M.E., 2003. Uptake of erythromycin by first-feeding sockeye salmon, Oncorhynchus nerka (Walbaum), fed live or freeze-dried enriched adult Artemia or medicated pellets. Journal of Fish Diseases 26, 277–285. Dixon, B.A., Van Poucke, S.O., Chair, M., Dehasque, M., Nelis, H.J., Sorgeloos, P., De Leenheer, A.P., 1995. COMMUNICATIONS: Bioencapsulation of the antibacterial drug sarafloxacin in nauplii of the brine shrimp Artemia franciscana. Journal of Aquatic Animal Health 7, 42–45. Duis, K., Hammer, C., Beveridge, M.C.M., Inglis, V., Braum, E., 1995. Delivery of quinolone antibacterials to turbot, Scophthalmus maximus (L.), via bioencapsulation: quantification and efficacy trial. Journal of Fish Diseases 18, 229–238. Finn, R.N., Rønnestad, I., van der Meeren, T., Fyhn, H.J., 2002. Fuel and metabolic scaling during the early life stages of Atlantic cod Gadus morhua. Marine Ecology-Progress Series 243, 217–234. Fukui, H., Fujihara, Y., Kano, T., 1987. In vitro and In vivo antibacterial activities of florfenicol, a new fluorinated analog of thiamphenicol, against fish pathogens. Fish Pathology 22, 201–207. Gapasin, R.S.J., Nelis, H.J., Chair, M., Sorgeloos, P., 1996. Drug assimilation in the tissue of European sea bass (Dicentrarchus labrax) fry delivered orally through bioencapsulation. Journal of Applied Ichthyology (Zeitschrift Fur Angewandte Ichthyologie) 12, 39–42. Gara, B., Shields, R.J., McEvoy, L., 1998. Feeding strategies to achieve correct metamorphosis of Atlantic halibut, Hippoglossus hippoglossus L., using enriched Artemia. Aquaculture Research 29, 935–948. Gaunt, P.S., 2004. Determination of dose rate of florfenicol in feed for control of mortality in channel catfish Ictalurus punctatus (Rafinesque) infected with Edwardsiella ictaluri, etiological agent of enteric septicemia. Journal of the World Aquaculture Society 35, 257–267. Gomez-Gil, B., Cabanillas-Ramos, J., Paez-Brambila, S., Roque, A., 2001. Standardization of the bioencapsulation of enrofloxacin and oxytetracycline in Artemia franciscana Kellogg, 1906. Aquaculture 196, 1–12.
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