A single‐dose pharmacokinetic study of emamectin benzoate in cod,Gadus morhua L., held in sea water at 9 °C

The pharmacokinetic profile of the antiparasitic agent emamectin benzoate was studied in plasma after intravenous (i.v.) injection and in plasma, muscle and skin following oral (p.o.) administration to cod, Gadus morhua, held in sea water at 9 °C and weighing 100–200 g. Following i.v. injection, the plasma drug concentration‐time profile showed two distinct phases. The plasma distribution half‐life (t_1/2α) was estimated as 2.5 h, the elimination half‐life (t_1/2β) as 216 h, the total body clearance (Cl_T) as 0.0059 L kg^?1 h^?1 and mean residence time (MRT) as 385 h. The volume of distribution at steady state, V_d(ss), was calculated to be 1.839 L kg^?1. Following p.o. administration the peak plasma concentration (C_max) was 15 ng mL^?1, the time to peak plasma concentration (T_max) was 89 h and t_1/2β was 180 h. The highest concentration in muscle (21 ng g^?1) was measured after 7 days and t_1/2β was calculated to be 247 h. For skin, a peak concentration of 28 ng g^?1 at 3 days was observed and a t_1/2β of 235 h was determined. The bioavailability following p.o. administration was calculated to be 38%.     

Multiple dose pharmacokinetic study of oxolinic acid in cod, Gadus morhua L.

The plasma, muscle and liver distribution and elimination of the antibacterial agent oxolinic acid were studied after multiple oral (p.o.) administration of 10 or 20 mg kg⁻¹ day⁻¹ to cod (Gadus morhua) for 6 days. The fish, held in seawater at 6 and 12°C and weighing 150–250 g were sampled 24 h following last medication. The concentrations in plasma and tissues were clearly dosage and temperature dependent. The distribution from plasma to muscle (muscle/plasma ratio) was higher than that from a single dose study and independent of temperature and dosage. The distribution from plasma to liver (liver/plasma ratio) was lower than the muscle/plasma ratio and according to this study dependent of the administered dosage but independent of temperature. The elimination of oxolinic acid from plasma, muscle and liver was considerably faster following multiple administration compared to a single administration.     

Immunochemical analyses of Vibrio anguillarum strains isolated from cod, Gadus morhua L., suffering from vibriosis

Abstract. Vibrio anguillarum isolates were sampled from cod fry showing clinical signs of vibriosis at different cod farms along the western coast of Norway. Except for one isolate, all were shown to cause mortality in laboratory challenge experiments. Using biochemical analysis and specific rabbit antisera (O1–O5), all the pathogenic isolates were classified as typical V. anguillarum serotype O2. SDS-PAGE and Western blot analysis of both whole bacterial cells and purified lipopolysaccharides (LPS) showed that the LPS structures were heterogeneous. ELISA analyses using adsorbed antisera identified the presence of two different subgroups within serogroup O2, which correspond with O2a/O2α and O2b/O2β described previously.     

Pharmacokinetics of oxolinic acid in gilthead sea bream, Sparus aurata L.

This is the first study on the pharmacokinetic parameters of oxolinic acid (OA) in gilthead sea bream, Sparus aurata L. The kinetic profile of OA was studied after a single intravascular injection (20 mg kg⁻¹) in 100 g fish at 20 °C. The distribution half-life ( t _1/2α) and the elimination half-life ( t _1/2β) of the drug were found to be short (0.51 and 12.60 h, respectively). The drug penetration from the plasma to the tissues was adequate as the apparent volume of distribution of the drug at steady-state ( V _d(ss)) was found to be 2.11 L kg⁻¹. The mean residence time ( MRT ) of OA was short (14.25 h) and the total clearance rate ( Cl _T) of the drug was low (0.15 L kg⁻¹ h⁻¹). The bioavailability ( F %) of OA following oral administration (30 mg kg⁻¹) was also low (14%). Maximum values were observed for muscle at 0.5 h after injection, with levels declining as with subsequent sampling. At the first two time points (0.5 and 1 h) plasma levels of OA were higher than muscle, however, the reverse was evident for subsequent samples. Following oral administration, highest muscle levels were found at 16 h and, with the exception of the 24-h sampling, muscle OA concentrations were higher than plasma at all time points. The fast elimination of OA suggests short withdrawal times with reference to human consumption of treated fish.     

A single-dose pharmacokinetic study of oxolinic acid and vetoquinol, an oxolinic acid ester, in Atlantic halibut, Hippoglossus hippoglossus L., held in sea water at 9 °C

The pharmacokinetic properties of the antibacterial agent oxolinic acid were studied after intravenous, intraperitoneal and oral administration to 1.5–3.0 kg Atlantic halibut, Hippoglossus hippoglossus L., held in sea water at 9 °C. Following intravenous injection, the plasma drug concentration-time profile showed two distinct phases. The terminal elimination half-life was estimated to be 52 h, whereas total body clearance (Cl_T) was determined to be 0.044 L kg^–1 h^–1. The volume of distribution at steady state, V_d(ss), was calculated to be 3.0 L kg^–1, indicating good tissue penetration of oxolinic acid in Atlantic halibut. The peak plasma concentration (C_max) and the time to peak plasma concentration (T_max) were estimated to be 1.2 and 2.7 μg mL^–1, and 21.5 and 80 h, respectively, following oral administration of medicated feed or intraperitoneal injection. The corresponding bioavailabilities were calculated to be 15% and 92%, respectively. Oral administration of vetoquinol, the carbitol ester of oxolinic acid, increased the bioavailability of oxolinic acid to 64% and the total bioavailability (oxolinic acid + vetoquinol) to 82%, whereas C_max and T_max values of 6.7 μg mL^–1 and 14.5 h, respectively, for oxolinic acid, and 1.0 μg mL^–1 and 6.3 h, respectively, for vetoquinol were obtained. Based on a minimum inhibitory concentration (MIC) of 0.0625 μg mL^–1 for susceptible strains, a single intraperitoneal injection of 25 mg kg^–1 of oxolinic acid maintains plasma levels in excess of 0.25 μg mL^–1, corresponding to four times the MIC value, for ≈12 days. The corresponding values for a single oral dose of 25 mg kg^–1 of oxolinic acid and vetoquinol were 5 and 10 days, respectively. For resistant strains with a MIC of 1 μg mL^–1, a single oral dose of vetoquinol (25 mg kg^–1) maintained plasma levels in excess of 4 μg mL^–1 for 34 h.     

Treating experimentally induced vibriosis (Listonella anguillarum) in cod, Gadus morhua L., with florfenicol

This study was performed to determine the efficacy of orally administered florfenicol in the treatment of experimentally induced vibriosis (Listonella anguillarum) in cod, Gadus morhua. The L. anguillarum strain HI-610 was used. This strain has a minimal inhibitory concentration value of 0.5 mg L(-1) against florfenicol. Fifteen groups of 40 fish each were challenged by bath with 1.7 x 10(5) CFU mL(-1) for 1 h. Three days following challenge, medication with florfenicol was introduced in 12 of the groups. The dosages used were 10 mg kg(-1) day(-1) for 10 consecutive days in marine or salmonid pellets, 10 mg kg(-1) day(-1) for five consecutive days in marine pellets or administered at days 1, 2, 4, 6 and 8 following initiation of treatment. Among challenged unmedicated fish mortality started at day 3 post-challenge reaching a final cumulative mortality of 77% at day 15. The experiment was terminated at day 26. In the medicated groups, the majority of deaths occurred from days 3-7 post-challenge reaching final cumulative mortalities of 31% and 52%, respectively, for the fish given marine and salmonid pellets for 10 consecutive days. The fish treated with medicated marine pellets for five consecutive days and at days 1, 2, 4, 6 and 8 (sequential feeding) following initiation of treatment had cumulative mortalities of 52% and 38%, respectively. Survival of medicated fish in all groups was significantly (P < 0.005) greater than survival of challenged unmedicated fish. Furthermore, a significant difference (P < 0.001) in survival was found between fish treated for 10 consecutive days using marine pellets and the groups using marine pellets for five consecutive days and salmonid pellets for 10 consecutive days. Twenty four hours following last medication, six fish had mean plasma concentrations of 3.3 +/- 1.7 and 3.5 +/- 2.8 microg mL(-1), respectively, in fish treated for 10 consecutive days using marine and salmonid pellets. Corresponding values for fish treated for five consecutive days and by sequential feeding were 2.2 +/- 2.3 and 1.7 +/- 0.7 microg mL(-1), respectively.     

Vaccination experiments and studies of the humoral immune responses in cod, Gadus morhua L., to four strains of monoclonal-defined Vibrio anguillarum

Abstract. Vibrio anguillarum isolated from diseased cod, Gadus morhua L., were serotyped by use of a panel of monoclonal antibodies. Four serotypes could be distinguished, having different lipopolysaccharide determinants. These phenotypic differences were also reflected in the genetic map, as revealed by fingerprinting of bacterial DNA. Antisera were raised in cod after immunization with the V. anguillarum serotypes, and Western blot techniques demonstrated production of specific antibodies mainly to LPS-antigens. The immune system in cod discriminates to a eertain degree between the four serotypes as shown by crossreactions of the immune sera in elisa . Moreover, it was also shown that natural antibodies to bacterial antigens are present in non-immune sera, but these specificities are non-LPS in nature. As a consequence of the heterogeneity of the V. anguillarum strains, vaccination experiments were performed under laboratory conditions to compare the effectiveness of bacterins based on either single vaccines or polyvaccines. The results from these experiments were promising since challenge with one strain demonstrated 100% protection both in fish vaccinated with the homologous serotype as well as a mixture of all the four serotypes.     

Temperature-dependent pharmacokinetics and tissue distribution of oxolinic acid in sea bass, Dicentrarchus labrax L., after a single intravascular injection

The pharmacokinetics and tissue distribution of oxolinic acid following an intravascular administration (15 mg kg^?1 fish) were determined in sea bass, Dicentrarchus labrax L. (110 g), at 13 °C and 22 °C water temperature. The kinetic profile of the drug was found to be temperature dependent, with increased temperature having a greater effect on distribution after equilibrium and the elimination phase than on the distribution process. The distibution half‐life of oxolinic acid was 1.15 and 2.76 h at 22 °C and 13 °C respectively, whereas the elimination half‐life of the drug was 55 h at 22 °C and 315 h at 13 °C. The values of the apparent volume of distribution (1.44 L kg^?1 at 22 °C and 3.31 L kg^?1 at 13 °C) and the volume of distribution at steady state (5.2 and 14.7 L kg^?1 at the high and low temperature respectively) were considerably different between the two tested temperatures. The total body clearance of the antibiotic was found to be low (1.47 L kg^?1 day^?1 at 22 °C and 0.80 L kg^?1 day^?1 at 13 °C). Lower rates of elimination were found for the liver compared with muscle, the difference increasing with increasing temperature, while elimination rates from the serum were higher than those of other tissues, especially at the high temperature.     

Treatment of vibriosis in European sea bass larvae, Dicentrarchus labrax L., with oxolinic acid administered by bath or through medicated nauplii of Artemia franciscana (Kellogg): efficacy and residual kinetics

European sea bass larvae were challenged by bath with Listonella anguillarum strain 332A, 2.5×10(7) CFUmL(-1) for 1h. Fish either received no treatment or oral treatment with Artemia franciscana (Kellog) nauplii enriched with oxolinic acid, or bath treatments with oxolinic acid. Medication commenced 1day following challenge and was performed on days 1, 3 and 5 post-challenge at a dosage of 20mgL(-1) for 2h for bath treatments, while two doses each of 750 nauplii per fish were administered daily for five consecutive days in oral treatments. Cumulative mortality reached 96% for the unmedicated challenged group, 32% in the group receiving bath treatments and 17% in the group receiving medicated nauplii. Pharmacokinetic parameters of oxolinic acid were calculated in sea bass larvae, for both treatments. Steady-state concentrations of oxolinic acid of 48.0 and 75.2μgg(-1) were achieved for bath treatment and oral treatment, respectively, while the elimination half-life was calculated to be 25.1h for bath treatment and 21.7h for oral treatment.     

An aerolysin-like enterotoxin from Vibrio splendidus may be involved in intestinal tract damage and mortalities in turbot, Scophthalmus maximus (L.), and cod, Gadus morhua L., larvae

Vibrio splendidus is a pathogen that can cause major losses during the early stages of larval turbot rearing when live feed (rotifers or Artemia) is used. As haemolytic bacteria have often been associated with larval rearing losses, we studied the role of the V. splendidus haemolysin in infection of larvae. From a bank of over 10,000 transposon mutants of V. splendidus, two different types of haemolysin-negative mutants were obtained. Both had lost virulence for larval fish, and immunohistochemistry showed that the transposon mutant studied colonized the turbot larval intestinal tract at a similar level to the wild-type organism but did not cause damage or signs of enteritis found with the wild-type organism. One transposon insertion site was located within a gene with high homology to aerolysin, the cytolytic toxin produced by several Aeromonas spp. The haemolysin, which we have termed vibrioaerolysin, had properties similar to aerolysin and osmotic protection studies showed that it formed pores in the membranes of erythrocytes of similar diameter to those of aerolysin. The Tn10 insertion site of the second transposon mutant was in an adjacent ToxR-like gene, suggesting that this might control expression of the vibrioaerolysin. The gastroenteritis caused by Aeromonas spp. in humans is considered to be due to production of aerolysin causing cyclic AMP-dependent chloride secretion in cells of the gastrointestinal tract. Damage to the intestinal tract of marine fish larvae could occur in a similar way, and it is possible that several Vibrio spp. found in the developing bacterial flora of the larval fish gut can secrete aerolysin-like toxins leading to death of larvae in the early rearing stages. Routine bacteriological screening on blood agar plates of live feed is recommended with measures to reduce the concentrations of haemolytic bacteria in rearing systems.