Detection of quinolone-resistance genes in Photobacterium damselae subsp. piscicida strains by targeting-induced local lesions in genomes

Quinolone-resistant strains of the fish-pathogenic bacterium, Photobacterium damselae subsp. piscicida are distributed widely in cultured yellowtail, Seriola quinqueradiata (Temminck & Schlegel), in Japan. The quinolone resistance-determining region (QRDR) was amplified with degenerate primers, followed by cassette ligation-mediated PCR. Open reading frames encoding proteins of 875 and 755 amino acid residues were detected in the gyrA and parC genes, respectively. Resistant strains of P. damselae subsp. piscicida carried a point mutation only in the gyrA QRDR leading to a Ser-to-Ile substitution at residue position 83. No amino acid alterations were discovered in the ParC sequence. A mutation in the gyrA gene was also detected in nalidixic acid-resistant mutants of strain SP96002 obtained from agar medium containing increased levels of quinolone. These results suggest that GyrA, as in other Gram-negative bacteria, is a target of quinolone in P. damselae subsp. piscicida. Furthermore, we attempted to detect a point mutation using targeting-induced local lesions in genomes (TILLING), which is a general strategy used for the detection of a variety of induced point mutations and naturally occurring polymorphisms. We developed a new detection method for the rapid and large-scale identification of quinolone-resistant strains of P. damselae subsp. piscicida using TILLING.     

Expression and characterization of the periplasmic cobalamin-binding protein of Photobacterium damselae subsp. piscicida

Cobalamin (vitamin B₁₂) is an essential cofactor in a variety of enzymatic reactions and most prokaryotes contain transport systems to import vitamin B₁₂. A gene coding for a periplasmic cobalamin-binding protein of Photobacterium damselae subsp. piscicida was identified by in silico analysis of sequences from a genomic library. The open reading frame was composed of 834 bp encoding a protein of 277 amino acids. The protein showed 61% identity with the vitamin B₁₂-binding protein precursor of P. profundum , 53% identity with the corresponding protein of Vibrio parahaemolyticus and 43% identity with the periplasmic binding protein BtuF of Escherichia coli. The expression of the native protein was investigated in P. damselae subsp. piscicida , but BtuF was weakly expressed under normal conditions. To characterize the BtuF of P. damselae subsp. piscicida , the recombinant protein was expressed with a C-terminal His₆-tag and purified; the molecular weight was estimated to be approximately 30 kDa. The protein does not contain any free thiol group, consistent with the view that the two cysteine residues are involved in a disulphide bond. The purified BtuF binds cyanocobalamin with an affinity constant of 6 ± 2 μ m .     

Blood chemistry and organ nutrient composition in Atlantic salmon, Salmo salar L., fed graded amounts of wheat starch

After feeding Atlantic salmon, Salmo salar L., five graded amounts of wheat starch from 0 to 310 g kg^?1, low but increased levels of glycogen in kidney, heart and gills were determined. No variations were found in proximate or glycogen compositions of muscle, whereas the liver composition reflected the diet composition. Whole-body homogenates varied in dry matter and lipid levels; the variation was according to g lipid eaten per fish. Plasma glucose levels ranged above average levels only when the starch level in the feed was higher than 220 g kg^?1. Although feed intake and thereby lipid intake increased as dietary carbohydrate increased in the present experiment, no differences in plasma concentrations of cholesterol and only small changes in triglycerides were determined. The increase in dietary carbohydrate was balanced with protein, and total plasma protein concentrations followed the decrease in feed protein content. Haematocrit, haemoglobin, mean cell volume and mean cell haemoglobin all showed significantly reduced levels as dietary starch increased, indicating a fibre effect from ‘left-over’ starch in the intestine followed by reduced absorption of divalent ions, such as iron.     

Flavobacterium psychrophilum in rainbow trout, Oncorhynchus mykiss (Walbaum), hatcheries: studies on broodstock, eggs, fry and environment

The occurrence of Flavobacterium psychrophilum at four rainbow trout hatcheries was investigated to provide more knowledge about the reservoirs and transmission of this bacterium. Broodstock were sampled at stripping (including both unfertilized and fertilized eggs), and the offspring were then sampled at the eyed egg and fry stages. Water and surface samples (e.g. hatchery trays) were also sampled. Flavobacterium psychrophilum was found in ovarian fluid and milt, indicating that broodstock may serve as a reservoir and are latent carriers of the pathogen. Flavobacterium psychrophilum was not found on or inside eggs, but further egg studies will be necessary to elucidate the possibility of vertical transmission of the pathogen. Flavobacterium psychrophilum was isolated from water samples, but only from water that had been in close contact with farmed rainbow trout or eggs. Flavobacterium psychrophilum isolates were characterized and compared with well-characterized strains, using degradation of elastin, serotype and ribotype profiles. Different ribotypes of F. psychrophilum were found between hatcheries, but a common ribotype A was found at all four hatcheries. Different ribotypes were found in broodstock without clinical disease, whereas only a few ribotypes (mostly ribotype A) were found in diseased fry. The same ribotype A was found in broodstock, in water samples from hatchery trays and in fry, which suggests the possibility of transmission of F. psychrophilum between broodstock and offspring.     

The essential fatty acid requirement of milkfish (Chanos chanos Forsskal)

The essential fatty acid (EFA) requirement of milkfish was examined by a 12-week feeding trial using defined, purified diets at water temperature of 28–29°C and salinity of 32. The test diets contained varying levels of 18:0 (triglyceride form, TG), 18:3(n–3), 18:2(n–6) and (n–3) highly unsaturated fatty acids (n–3 HUFA). Milkfish juveniles were starved for 7 days and were than fed lipid-free diet for 30 days before the initiation of feeding trials. Low growth and feed efficiency together with high mortalities were observed in fish fed the lipid-free diet as well as in the EFA-deficient diet. Supplementation of 2% 18:2(n–6) to the tristearin based diet did not improve growth rate of milkfish as effectively as feeding with (n–3) fatty acids. The highest weight gain was obtained in milkfish fed a combination of 5% 18:0 + 1.0% 18:3(n–3) + 0.5% 20:5(n–3) + 0.5% 22:6(n–3) although the supplementation of 2% 18:3(n–3) alone or combination of 0.5% 20:5(n–3) + 0.5% 22:6(n–3) to the tristearin based diets were also effective for improvement of growth. Thus, (n–3) fatty acids, such as 18:3(n–3) and (n–3)HUFA were nutritionally more important than 18:2(n–6) for milkfish. The fatty acid composition of the polar lipids from whole body of milkfish juveniles fed the various test diets were influenced by the composition of the dietary fatty acids.     

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.     

Effects of stocking density on the survival, growth, size variation and condition index of juvenile rabbitfish Siganus rivulatus

Spinefoot rabbitfish, Siganus rivulatus, is an economically important species of herbivorous fish that is relatively easy to rear and thus considered to be suitable for aquaculture. Juveniles are generally reared in nursery systems before being stocked into growout cages or ponds. We report here our evaluation of the effects of stocking density on the survival, growth, feed efficiency and condition index of S. rivulatus juveniles in nursery tanks. The experiment was conducted in a recirculating system of twelve 52-l aquaria connected to a biological filter and a sand filter. Juvenile fish (average weight 6.5 g) were stocked into aquaria at four stocking densities (10, 20, 30, and 40 fish/aquarium) with three replicate aquaria per treatment. Diet was provided at 3% body weight daily divided into two feedings. Fish were weighed weekly for 8 weeks and the diet increased accordingly. Survival was greater than 95% in all treatments, with no significant differences observed among treatments. There were also no differences in specific growth rate (SGR 2.12–2.27) of the fish among treatments. Growth rate was linear during the 8 weeks in all treatments, and harvested biomass increased proportionally to stocking density (198, 401, 600 and 785 g per increasing stocking density, respectively). Feed efficiency (FE 0.67–0.71) of the fish did not vary significantly among treatments. The coefficient of variation was high (35–41%) among the harvested fish, but it also did not differ significantly among treatments. The final condition indices of the fish in all treatments were similar to each other but significantly greater than the initial values (P < 0.05). The results suggest that there is no apparent effect of stocking density at the levels tested on the survival and growth of juvenile rabbitfish.     

An assessment of salmon farms and wild salmonids as sources of Lepeophtheirus salmonis (Krøyer) copepodids in the water column in Loch Torridon, Scotland

Wild salmonids and farmed salmon can both be sources of Lepeophtheirus salmonis (Krøyer, 1838) larvae . Farmed salmon smolts free of L. salmonis infections are stocked in sea cages and may subsequently contract L. salmonis infections, probably from wild fish. The contribution of gravid L. salmonis at Atlantic salmon, Salmo salar L., farms to populations of L. salmonis larvae in the water column has in the past been based on estimated parameters, such as louse fecundity. This present study augments these calculations by combining empirical data on densities of infective L. salmonis copepodids in the field with estimates of the number of gravid L. salmonis on farmed and wild salmonids in Loch Torridon. Data collected between 2002 and 2007 show a significant correlation between mean densities of L. salmonis copepodids recovered in the water column and the numbers of gravid L. salmonis at the local salmon farms. Generally, the farms with greatest numbers of salmon were observed to have stronger correlations with densities of copepodids in the water than the farms with fewer fish. The study suggests that louse management approaches, e.g. treatment trigger levels, need to take account of individual farm biomass, or numbers of fish. This study highlights the importance of control of L. salmonis on salmon farms for the co-existence of both wild salmonid populations and the aquaculture industry.     

Beneficial properties of lactic acid bacteria isolated from a Rana catesbeiana hatchery

This work addresses the selection of potentially probiotic lactic acid bacteria (LAB) to be used in raniculture. Thus, strains belonging to the genera Pediococcus pentosaceus, Leuconostoc mesenteroides, Lactococcus lactis and Enterococcus faecium isolated from a Rana catesbeiana hatchery were evaluated for their inhibitory properties against RLS-associated pathogens ( Proteus vulgaris, Pseudomonas aeruginosa, Staphylococcus epidermidis ) and food-borne bacteria. Cell-free supernatants of LAB strains inhibited the growth of at least one of the pathogens by organic acids, but L. lactis CRL 1584 also produced a bacteriocin-like metabolite. The ability of LAB strains to produce H₂O₂ in MRS+TMB medium was also studied. Seventy-eight to ninety six per cent of the strains showed some level of H₂O₂ production. Moreover, different organic solvents were used to determine the hydrophobicity and Lewis acid/base characteristic of LAB strain surfaces. Most of the strains presented hydrophilic properties, but no acidic or basic surface characters. However, some strains isolated from the skin showed a high degree of hydrophobicity and basic components in the cell surface due to their adhesion to chloroform. These properties were not observed in LAB from balanced feed and freshwater. Taking into account general guidelines and the beneficial properties studied, five strains were selected as potential candidates to be included in a probiotic for raniculture.