George Rigos

Antibacterial Agents in Mediterranean Finfish Farming: A Synopsis of Drug Pharmacokinetics in Important Euryhaline Fish Species and Possible Environmental Implications

The literature pertaining to the use of registered antibacterial agents in Mediterranean finfish farming is reviewed, with an emphasis on the Greek fish-farming industry. This review provides a scientific resource dedicated to the design of future antibacterial dosing regimes in Mediterranean fish farming, where insufficient supporting information is currently available. This paper addresses the paucity in knowledge concerning pharmacokinetics and the efficacy and environmental impact of commonly used antibacterials needed to direct future research and promote good practices in the euryhaline fish farming industry. Several registered antibacterials are currently available for combating bacterial infections, including tetracyclines, (fluoro) quinolones, potentiated sulfa, penicillin and chloramphenicol derivatives. Based on the available data, oxytetracycline (OTC) and quinolone drugs (oxolinic acid – OA and flumequine – FLU) are the most widely used in Mediterranean aquaculture. As a result these drugs have received the most extensive studies, whereas, there is considerable paucity of reliable data on pharmacokinetic and the depletion characteristics of other drugs used, particularly potentiated sulfa, penicillin derivatives and florfenicol. We find there is incomplete data on drug efficacy and minimum inhibitory concentrations (MIC) for common antibacterials used against the major bacterial pathogens of Mediterranean fish species. Furthermore, a considerable lack of data on environmental drug concentrations around Mediterranean fish farms was also identified, highlighting the need for more extensive environmental studies to monitor contamination in environmental components i.e., water and sediment, and in non-target species (flora and fauna). Prudent selection and use of antibacterials can encourage lower dosage applications, enhance treatment efficacy, and help to minimize contamination of the environment. Selection of readily bioavailable drugs which have low environmental persistence, low aquatic toxicity and high antibacterial efficacy is advised, to reduce potential losses to the environment and associated toxic effects on target species and the development of bacterial resistance. Lack of present data made it impossible to provide thorough and accurate guidance on selection and use of antibacterials and approaches for minimizing environmental impacts for the treatment of major euryhaline aquaculture species.

Pharmacokinetics and tissue distribution of oxytetracycline in sea bass, Dicentrarchus labrax, at two water temperatures

Abstract A pharmacokinetic study of oxytetracycline (OTC) following an intravascular administration (40 mg/kg) was carried out in sea bass, Dicentrarchus labrax (110 g), at 13.5 and 22 °C water temperature. Blood, muscle and liver samples were taken at 1, 2, 4, 8, 16, 32, 64 and 128 h post-injection. The plasma data were conformed to a two-compartment model. The kinetic profile of the drug was found to be temperature dependent. The absorption half-life (t1/2α) of OTC was 0.98 and 0.192 h at 13.5 and 22 °C, respectively, whereas the elimination half-time (t1/2β) of the drug was 69 h at 13.5 °C and 9.65 h at 22 °C. The apparent volume of distribution of the drug at steady state [Vd(ss)] was 5.62 l/kg at 13.5 °C and 2.59 l/kg at 22 °C. The mean residence time (MRT) of OTC was found to be 37.7 h at 22 °C and 71 h at 13.5 °C. The total clearance of the drug (CLT) was calculated to be 73.5 and 68.7 ml/kg/h at 13.5 and 22 °C, respectively. Liver levels indicated higher OTC values than respective muscle levels at all time points and for both temperatures. The elimination of OTC from tissues tested was faster at the high temperature, whereas the drug was eliminated faster from liver compared to muscle when comparisons are made at the same temperature.

Pathological obstacles of newly-introduced fish species in Mediterranean mariculture: a review

Species diversification has been extensively employed in Mediterranean mariculture industry as a tool to relief the crisis resulting from market saturation and overproduction of the two main representatives, sea bass (Dicentrarchus labrax) and gilthead sea bream (Sparus aurata). Numerous new promising candidates belonging to several fish families such as Sparidae, Polyprionidae, Serranidae, Sciaenidae, Soleidae, Carangidae and Scombridae have been introduced to provide alternative culture choices in Mediterranean. The farming potential of these species in production scale has received some or little success partly due to the fact that their entrance has not been accompanied without the presence of considerable pathological problems. Bacterial outbreaks causing serious problems have been mainly induced by Photobacterium damsella subsp. piscicida, Vibrio alginolyticus, V. harveyi, Tenacibaculum maritimum and Flavobacterium sp. Detrimental parasitic pathogens included the myxosporean Enteromyxum leei, the ciliates Cryptocaryon irritans and Uronema spp., the flagellate Amyloodinium occelatum, the monogenean Zeuxapta seriolae and the digeneans Paradeontacylix-like spp. Viral encephalopathy and retinopathy infections and the chlamydia-like bacteria causing Epitheliocystis disease are also of significance importance. Numerous other agents of lesser pathogenic significance as well as other occasionally recorded pathogens are also mentioned in this review. Additional research effort has to be devoted to the pathogenesis of important pathogens and the interactions with their hosts to assist the viability of new fish species farming in Mediterranean region.

Pharmacokinetics and bioavailability of oxytetracycline in gilthead sea bream (Sparus aurata) after a single dose

Abstract This is the first study to investigate the pharmacokinetic parameters of oxytetracycline (OTC) in gilthead sea bream ( Sparus aurata ). Following a single intravascular injection of OTC (40 mg/kg) in 100 g fish kept at 20 °C, the distribution half-life ( t 1/2 α ) and the elimination half-life ( t 1/2 β ) of OTC were found to be long (2 and 53 h, respectively). The drug was well distributed throughout the internal body compartments since the apparent volume of distribution of the drug at steady-state ( V d(ss) ) and the apparent volume of distribution by the area method ( V d(area) ) were found to be high (2.9 and 3.8 l/kg, respectively). OTC had a long mean residence time (MRT) (56 h) but a short total clearance (CL T ) (0.05 l/kg h). The slow elimination of the drug suggests a need for long withdrawal periods prior to use of dosed fish for human consumption. Although the bioavailability ( F %) of OTC following oral administration in aqueous suspension (75 mg/kg) was calculated to be low (9%), maximum OTC plasma concentration was relatively high (2.5 μg/ml), indicating that current therapeutic regimens applied to gilthead sea bream might be effective against bacterial pathogens.

Myxosporean Infections in Greek Mariculture

Aquaculture in Greece has expanded rapidly in the last decade, mainly with the involvement of the intensive farming of euryhaline fish species. This rapid development has been inevitably accompanied by an increased occurrence of different pathogens, including Myxosporea, creating a considerable constraint on the further development of the industry. Recently, significant problems due to myxosporean infections in Mediterranean mariculture have emerged (Diamant, 1992; Le-Breton and Marques, 1995; Athanassopoulou et al., 1998). The present work describes the main myxosporean pathogens occurring in Greek cage culture from September 1996 to September 1997. The potential importance of these parasites is discussed along with some epidemiological data. Fish samples from 30 cage farms from seven geographical regions were examined whenever mortalities were evident. Sea bass, Dicentrarchus labrax L., gilthead sea bream, Sparus aurata L., sharpsnout sea bream, Puntazzo puntazzo (Cetti) and common dentex, Dentex dentex L. were associated with 42, 29, 9 and 2 mortality incidences respectively. At least 10 moribund individuals were examined from each case. Fresh smears of gills, skin, gut, gall bladder and kidney were examined. Tissues of gut, kidney, heart, spleen, gills and liver were fixed in 10% buffered formalin and processed for paraffin histology. Sections (5 mm) were stained with haematoxylin and eosin and examined under light microscopy. Myxosporeans were identified according to Lom and Arthur (1989). Myxosporeans were found in almost 40% of the examined cases, on some occasions associated with considerable fish losses. The culture of sharpsnout sea bream seemed to be completely inhibited by Myxidium leei Diamant et al., 1994 suffering significant (1–5% day) and prolonged mortalities (3–6 weeks). On some occasions, 30–70% of the population was lost. Maximum mortalities were evident by

Tissue distribution and residue depletion of oxolinic acid in gilthead sea bream (Sparus aurata) and sharpsnout sea bream (Diplodus puntazzo) following multiple in-feed dosing

Abstract The tissue distribution and residue depletion of oxolinic acid (OA) were investigated in gilthead sea bream and sharpsnout sea bream after a multiple (10 days) in-feed administration (30 mg/kg fish) at 19 °C. Plasma, muscle, skin, liver and bile were sampled daily during treatment (1–10 days) and post-treatment (11,12, 14, 15 and 17 days) and subsequently analysed for OA concentrations using high-performance liquid chromatography and fluorescent detection (HPLC-SFD). Oxolinic acid displayed biphasic elimination in tissues of both species during treatment and reached “consumer safe levels” 24 h after treatment. OA concentration in gilthead sea bream peaked on day 4, with highest concentrations in liver (2660 ng/g), followed by bile (2370 ng/ml), skin (2350 ng/g), muscle (1330 ng/g) and plasma (880 ng/ml). In sharpsnout sea bream, peaked OA concentrations were observed in liver (2720 ng/g) on day 4, while highest concentrations were measured later on day 5 in plasma (910 ng/ml) and day 6 in bile (2360 ng/ml), skin (1960 ng/g) and muscle (1770 ng/g). The calculated areas under the concentration curves (AUC) also reflect a good tissue distribution of OA. Elimination half-lives ( t 1/2 ) were estimated to be 11–14 and 11–19 h in tissues of gilthead sea bream and sharpsnout sea bream, respectively. Multiple dosing of OA in sparids should be adequate to confront bacterial pathogens considering published minimum inhibitory concentrations of the drug.

Effect of ivermectin on the liver of gilthead sea bream Sparus aurata: A proteomic approach

Gilthead sea bream Sparus aurata is the most commercialized Mediterranean aquacultured fish species. Ivermectin has recently (experimentally) started to be used to control ectoparasitic infestations in Mediterranean cultured marine fish. The potential hepatotoxicity of ivermectin was investigated in gilthead sea bream juveniles (35g) following oral administration at the recommended dose of 0.2 mgkg(-1) fish for 10d. Difference Gel Electrophoresis Technology (DIGE) was used to study the effect of this treatment in gilthead sea bream liver protein profile under routine culture conditions. The 2D-DIGE protein maps obtained were analyzed using the DeCyder 6.5 software. The results obtained showed significant changes in the expression of 36 proteins respect to the control group. Among these proteins, six increased in abundance, and 30 decreased. Spot showing differential expression respect to the control were analyzed by mass spectrometry and database search, which resulted in three positive identifications corresponding to hepatic proteins involved in lipid metabolism (apoA-I), oxidative stress responses and energy generation (beta-globin, ATP synthase subunit beta). These proteins have not been previously associated to invermectin effect.

Effects of graded dietary levels of soy protein concentrate supplemented with methionine and phosphate on the immune and antioxidant responses of gilthead sea bream (Sparus aurata L.)

ABSTRACT The effects of a dietary soy protein concentrate (SPC) as a fish meal (FM) substitute, on selected innate immune responses, the oxidative status, hepatic and intestinal morphology of gilthead sea bream, Sparus aurata, were evaluated after a three‐month feeding trial. Isonitrogenous (45% crude protein) and isoenergetic (23 kJ/g gross energy) diets with 20% (SPC20), 40% (SPC40) and 60% (SPC60) of SPC inclusion, supplemented with methionine and phosphate, were evaluated against a diet containing FM as the sole protein source. Diets were allocated in triplicate groups of 26‐g fish (8 kg m−3/tank) and administered for three months. Immune responses were evaluated by performing immunological assays in blood (respiratory burst activity) and serum (myeloperoxidase content, bacteriolytic and lysozyme activity), as well as by gene expression analysis of immune‐associated genes (MHCII&agr;, &bgr;2m, CSF‐1R, NCCRP‐1, TGF‐&bgr;1, HSP70) in the head kidney and distal intestine. In addition, oxidative stress was evaluated by measuring the activity of liver enzymes associated with the antioxidant system. The respiratory burst activity of blood was significantly decreased in the SPC40 group, while serum myeloperoxidase content and bacteriolytic and lysozyme activities were affected. Significantly higher expression levels of NCCRP‐1 and HSP70 were found in SPC60 head kidneys, while increased intestinal MHCII&agr; and NCCRP‐1 transcripts were observed in SPC40. Hepatic antioxidant enzyme activity of glutathione reductase and glutathione‐S‐transferase was significantly enhanced in the SPC40 and SPC60 groups, while superoxide dismutase activity was increased only in the SPC40 group. Moreover, increased lipid accumulation in the enterocytes of the distal intestine was observed in the SPC60 group. Overall, a three‐month feeding period with diets over 40% of dietary SPC inclusion as a FM substitute, indicated increases on immune and antioxidant enzyme responses, suggesting the dietary SPC levels that gilthead sea bream can tolerate. HIGHLIGHTSInclusion of dietary SPC exhibited immunomodulatory effects for gilthead sea bream above 40%.Moderate morphological alterations were observed in the distal intestine at 60% level of SPC.Above 40% of SPC inclusion the activity of antioxidant enzymes was increased.SPC inclusion up to 40% was suggested as the tolerance dietary levels for gilthead sea bream.

Growth performance and product quality of meagre (Argyrosomus regius) fed diets of different protein/lipid levels at industrial scale

Abstract The rearing of meagre (Argyrosomus regius) up to commercial sizes, using diets of different protein/lipid ratios, was examined in two long-term trials. In the first 2 × 2 trial, four diets containing two protein (43% and 47%) and two lipid levels (15% and 20%) were evaluated in fish of 350 g initial weight. Fish were reared for 8 months in triplicate experimental cages, up to final weight of 900g. Growth performance showed that diets containing 43% protein were the most appropriate, as indicated by the better thermal growth coefficient (TGC): 0.70 and 0.71 (vs. 0.61 and 0.53 for 47/15 and 47/20 diets) and better daily growth index DGI: 0.91 and 1.00 (vs. 0.88 and 0.79 for 47/15 and 47/20, respectively). Liver fat did not differ among groups, but liver glycogen in the 43/15 dietary group was found to be significantly higher than the 47/20 (3.89% vs 1.88%). The histological examinations revealed a trend for increased lipid deposition when fish were fed high fat diets. Diets that performed best, namely 43/15 and 43/20, were used in a second trial conducted at a commercial fish farm. Fish weighing 520g were reared for 7 months up to 1100 g final weight. No significant differences were observed in the growth parameters examined. The feed conversion ratio was found to be better in the 43/20 diet compared to the 43/15 (1.58 vs. 1.68, respectively). The dietary fat levels significantly affected fillet fat content. However, such difference in nutritional content was not reflected in human-perceived sensory differences.