Exophiala angulospora causes systemic inflammation in atlantic cod Gadus morhua

Species of Exophiala are opportunistic fungal pathogens that may infect a broad range of warm- and cold-blooded animals, including salmonids and Atlantic cod. In the present study, we observed abnormal swimming behaviour and skin pigmentation and increased mortality in cod kept in an indoor tank. Necropsy revealed foci of different sizes with a greyish to brownish colour in internal organs of diseased fish. The foci consisted of ramifying darkly pigmented fungal hyphae surrounded by distinct layers of inflammatory cells, including macrophage-like cells. In the inner layer with many hyphae, the macrophage-like cells were dead. We observed no apparent restriction of fungal growth by the inflammatory response. A darkly pigmented fungus was repeatedly isolated in pure culture from foci of diseased fish and identified as Exophiala angulospora using morphological and molecular characters. This species has not been previously reported to cause disease in cod, but has been reported as an opportunistic pathogen of both marine and freshwater fish. Based on the morphology and sequence analysis presented here, we conclude that E. angulospora caused the observed chronic multifocal inflammation in internal organs of cod, leading to severe disease and mortality.     

Immunohistochemistry of Atlantic cod larvae Gadus morhua experimentally challenged with Vibrio anguillarum

Farming of Atlantic cod Gadus morhua is one of the most rapidly growing sectors of Norwegian aquaculture. Classical vibriosis caused by Vibrio anguillarum is a problem in cod aquaculture. To prevent disease outbreaks, a thorough understanding of the infection route and the impact of the bacteria on the host is important. The intestinal tract, skin and gills have all been proposed as routes of entry for bacterial infections such as vibriosis. We aimed to further develop understanding of V anguillarum serotype O2alpha infections in cod larvae by elucidation of a possible route of entry, the pattern of infection and its histopathology. Cod eggs were transferred to a 24-well polystyrene multi-dish with 2 ml of sterile aerated 80% (28 per thousand salinity) seawater. Challenge doses were 10(4) and 10(6) CFU ml(-1). Unchallenged larvae were used as controls. Larvae for immunohistochemical examination were sampled daily from each group. In most of the larvae, either no or very few bacteria were observed. Typical findings were clusters of bacteria in the spaces between the primary gill lamellae. None of these bacteria seemed to have adhered to the gills. Intestines of 3 out of 161 larvae examined contained positively immunostained bacteria. Some bacteria appeared attached to the microvilli, but none was observed inside epithelial cells. Only 2 larvae from the low-challenge dose group showed clear signs of histopathology, which occurred in the intestine. It is not possible to draw any conclusions regarding the portal of entry.     

Intraperitoneal vaccination of Atlantic cod, Gadus morhua with heat-killed Listonella anguillarum enhances serum antibacterial activity and expression of immune response genes

Serum-mediated reduction in bacterial count and expression of a number of immune response genes in the blood of Atlantic cod, Gadus morhua were investigated following intraperitoneal vaccination with heat-killed Listonella (Vibrio) anguillarum. Blood was collected from the caudal vein of both vaccinated and non-vaccinated (PBS-injected) fish at 0, 1, 3, 7 and 10 days post-vaccination (dpv). Serum protein concentration and antibacterial activity of the serum samples were determined. Whole blood was used for semi-quantitative RT-PCR of immune-related genes. Total serum protein was not significantly different between the vaccinated and non-vaccinated groups. Sera from the vaccinated fish significantly reduced L. anguillarum count on 3 dpv, with reductions of at least 2 log colony forming units per ml (CFU/ml) relative to the non-vaccinated fish. Expression of antibacterial genes, bactericidal/permeability-increasing protein/lipopolysaccharide-binding protein (BPI/LBP), g-type lysozyme and transferrin was significantly upregulated in the vaccinated fish, with maximum expression within 7 dpv. Cytotoxic-related and cell-mediated immunity genes such as, apolipoprotein A-I and the non-specific cytotoxic cell receptor protein (NCCRP-1) had maximum expression at 3 and 7 dpv, respectively. Significant upregulation in expression of pro-inflammatory cytokines, IL-1 beta and IL-8 was also observed in the vaccinated fish at 1 dpv. The upregulation of immune response genes following vaccination provides valuable information in the understanding of immune mechanisms against vibriosis in Atlantic cod particularly on the acute phase response during bacterial infection.     

Sympathetic nervous control of the iris sphincter of the atlantic cod,Gadus morhua

Summary The autonomic nervous control of the cod iris has been studied. The pharmacological properties of the smooth muscles of the iris have been elucidated by agonist/antagonist studies on isolated strip preparations. Electrical stimulation of parasympathetic and sympathetic pathways to the eye have been carried out, with recordings of the movements of the iris margin. Additions of cholinergic and adrenergic antagonists in selective concentrations were made to investigate the nature of the autonomic nerve fibres controlling the iris.Isolated strip preparations of the iris sphincter contracted in response to cholinergic or-adrenoceptor agonists. There appear to be no radial muscular elements in the cod iris. The effect of carbachol on the iris sphincter could be competitively antagonized by atropine, suggesting the presence of muscarinic receptors of the smooth muscles. The effect of adrenaline was similarly antagonized by phentolamine. The effect of phentolamine, and the order of potency for the adrenergic agonists, shows the presence of-adrenoceptors in the iris sphincter.-adrenoceptors of minor importance are also suggested by the inhibitory effects of isoprenaline on preparations pre-contracted by carbachol.The indirectly acting adrenergic agonist tyramine also contracts the isolated sphincter preparations. This effect is probably due to release of nervously stored catecholamines, since tyramine lacks effect on preparations from animals pre-treated with 6-hydroxydopamine. Preparations from 6-hydroxydopamine pre-treated animals also show a 10-fold increase in the affinity for adrenaline, demonstrating the development of a pre-synaptic supersensitivity due to the destruction of adrenergic nerve terminals of the iris. Stimulation of the sympathetic chain or ciliary nerves produces a constriction of the pupil of the same side. Application of selective concentrations of the antagonists atropine and phentolamine shows that the sympathetic constrictory innervation is solely adrenergic. In some preparations a small pupillo-dilatory effect of nerve stimulation is evident after the constrictory effect has been abolished by phentolamine. This inhibitory effect can be abolished by propranolol, indicating the presence of a-adrenoceptor mediated inhibitory control of minor importance. Stimulation of the oculomotor nerve produces no consistent responses of the cod iris.Illumination of one eye produces a pupilloconstriction comparable to that seen after sympathetic nerve stimulation. The light induced response is insensitive to atropine, phentolamine and tetrodotoxin, showing a direct effect on the smooth muscles of the sphincter. There is no consensual reflex in the cod.I wish to thank Dr. Susanne Holmgren for critically examining the original draft of this paper, and Mrs. Lena Utter for skilled assistance with isolated strip preparations and processing of concentration-response data. The fish was kindly supplied by Mr. Ingmar Hakemar. This work has been supported by grants from the Swedish Natural Science Research Council, the M. Bergvall Foundation and the Adlerbert Foundation.     

The role of spawning stock in formation of recruitment of northeast atlantic cod Gadus morhua

On the basis of statistical treatment of the data for 1946–2003 (correlation analysis and analysis of variance) on the biomass of the spawning stock (SSB) and of the resulting recruitment (R), expressed as abundance of three-year-old fish (N₃), of the Northeast Atlantic cod Gadus morhua, the relationship SSB → N₃ is revised and the extent of the influence of SSB on the formation of R is assessed. The relationship of SSB → N₃ turned out to be significant only at SSB ≤ 250 000 t. The influence of SSB is only 4.3–14.7%, in comparison to the other background factors controlling R. This casts doubt on the adequacy of the so-called precautionary approach in fishery management, popular in international fishery organizations, which is based on efforts to support the spawning stock at a certain level (B_pa) taken as the optimum one.     

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

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

Stages of embryonic development in the Atlantic cod Gadus morhua

The early development of the Atlantic cod, Gadus morhua was studied from fertilization until first-feeding. Multiple families were reared at 7 degrees C and a developmental staging series was prepared using morphological landmarks visible with the light microscope. Stages were named rather than numbered to allow for future additions and broadly grouped into larger time intervals called periods. The most useful staging features were found to be initially cell number, and later in development, somite number. The mean cell cycle time for the first six cleavages was 135 min and the linear regression equation for development of somites(s) over time (t) was s = 0.29t - 18.14. The segmentation period began at 220 h postfertilization (hpf), and unlike some other teleosts, the addition of new somites continued throughout the majority of embryonic development, until just prior to hatching. Hatching occurred at 256 hpf, after which individuals remained motionless at the water's surface, undergoing negative phototaxis only after the first day posthatch. The first-feeding stage was reached at the end of the third day posthatch, subsequent to development of a functional jaw and hindgut. This staging series provides an essential baseline reference for future experiments involving developing cod embryos and for the aquaculture industry.     

Visual contrast thresholds in the cod Gadus morhua L.

The contrast discrimination of Gadus morhua L. was studied by means of a cardiac conditioning technique. Fish were trained to respond to a projected pattern of spots and the contrast of pattern and background then reduced until the minimum contrast required to elicit a response was established. This was repeated at seven different background light levels to cover the range of light conditions naturally encountered by the cod. The minimum detectable contrast decreased with increasing light level to a minimum of around 2.0%. The contrast threshold curve showed a discontinuity at a light level of approximately 8.0 × 10^-6 W sr^-1 m^-2. This was thought to be linked to the change from photopic to scotopic vision. Significant differences were found in cod taken from two different locations. Overall, cod contrast detection compared very favourably with figures available for other species including man. Using the obtained contrast discrimination figures some estimates were made of the cod sighting distance of hypothetical targets under natural conditions.     

Antigen uptake and immunoglobulin production in Atlantic cod (Gadus morhua L.) after intraperitoneal injection of Vibrio anguillarum

Atlantic cod (Gadus morhua L.) were injected intraperitoneally with formalin-killed Vibrio anguillarum bacteria. Immunostaining revealed uptake of V. anguillarum antigens especially in the spleen after intraperitoneal (i.p.) administration. The uptake was time dependent in the interval 1-24 h. Most of the antigen uptake in the spleen was concentrated in areas around small blood vessels, while immunoglobulin producing cells were localised to some thick walled arteries. There was apparently little or no co-localisation of antigens and antibody producing cells. In the heart, some of the high endocardial endothelial cells of the atrium contained bacterial antigens and in head kidney some macrophage-like cells were stained. Very little antigen was found in the pigmented loose connective tissues of the peritoneum. In contrast, endothelial cells of the underlying blood vessels contained substantial amounts. In the heart, peritoneum and anterior kidney the number of antigen positive cells did not seem to change in the time interval 1-24 h. After i.p. immunisation with a mixture of V. anguillarum and Freunds complete adjuvant, the humoral immune response in Atlantic cod was low when tested 21, 42 and 105 days later. There was apparently no enhanced number of immunoglobulin synthesising cells caused by the antigen stimulation.     

Neurotransmitters in the intestine of the Atlantic cod, Gadus morhua

The effects of the putative neurotransmitters acetylcholine, adrenaline, adenosine, ATP, bombesin, 5-hydroxytryptamine, met-enkephalin, neurotensin, somatostatin, substance P and VIP have been investigated in the perfused intestine of the cod, Gadus morhua. The presence and distribution of the different types of nerves was investigated with immunohistochemistry and Falck-Hillarp fluorescence histochemistry. A spontaneous rhythmic activity of the perfused preparations usually occurred within a few minutes from the start of the experiment. This activity was diminished or abolished by addition of atropine, methysergide or tetrodotoxin to the perfusion fluid. Acetylcholine, 5-hydroxytryptamine or substance P caused a contraction of the intestinal wall. The response to acetylcholine was blocked by atropine but not by tetrodotoxin, while the response to 5-hydroxytryptamine was blocked by methysergide and usually also by tetrodotoxin. This indicates that the effect of acetylcholine is direct on the muscle cells, while the effect of 5-hydroxytryptamine may be at least partly via a second neuron. All adrenergic agonists (adrenaline, isoprenaline and phenylephrine) had a dominating inhibitory effect on the intestine. Experiments with antagonists showed that the inhibition is due to stimulation of both alpha-adrenoceptors and beta-adrenoceptors. ATP, adenosine and somatostatin also caused a relaxation of the intestinal wall, often followed by a contraction. Met-enkephalin produced variable responses, either a relaxation, a contraction or both. Bombesin caused a weak inhibition, if anything. Neurotensin and VIP did not visibly affect the intestinal motility. 5-HT-, substance P- and VIP-like immunoreactivity and catecholamine fluorescence were observed in the myenteric plexus, submucosa and muscle layers in all parts of the intestine.(ABSTRACT TRUNCATED AT 250 WORDS)     

Spectral sensitivity of the cod,Gadus morhua L.

The spectral sensitivity of the cod was determined under both dark adapted and light adapted conditions in the laboratory. Cod were trained by cardiac conditioning to detect a difference in radiance between an image of spots and the background radiance of a screen. Thresholds for this response were measured for a range of different wavelengths, and expressed as quantum adjusted values. Electroretino‐graphic studies were also performed on the eyes of cod, and spectral sensitivity curves prepared. Under dark adapted conditions both the behavioural and e.r.g. derived curves showed greatest sensitivity in the blue/green at 490 nm, matching the absorption curve for rhodopsin. A secondary peak in the behaviourally derived curve in the green/yellow at 550 nm indicated that a population of yellow cones may be implicated with the rods in scotopic vision. Under light adapted conditions the behavioural curves showed a shift to the blue, perhaps indicating an adaption to the high red content of the illuminating source. The e.r.g. curve showed greatest sensitivity to blue/green, as in the scotopic experiments but with an enhanced response at 550 nm, indicating greater cone activity. It is suggested that there is complex interaction between rods and cones in the cod retina, both types of receptor being active over a wide range of light intensities.     

Identification of transferrin in Atlantic cod Gadus morhua epidermal mucus

The previously unreported presence of transferrin in Atlantic cod Gadus morhua epidermal mucus is described. A less destructive sampling method, which may result in decreased epidermal tissue damage, is discussed.     

Haemoglobin polymorphism in atlantic cod(Gadus morhua): Allele frequency variation between yearclasses in a Norwegian fjord stock

A total of 262 specimens (0-, and 1-group) of Atlantic cod(Gadus morhua) representing 4 different yearclasses were caught in Trondheimsfjorden, Norway, during 1977–1984. They were genotyped by agar gel electrophoresis for the polymorphic haemoglobin locusHbI (Sick, 1961). The analyses revealed a highly significant (P=0.0003) heterogeneity ofHbI allele frequencies between yearclasses. The difference in the frequency of theHbI-1 allele between the first (1977) and the last (1983) yearclass amounted to 0.18 (±0.07). The results appear to support recent reports on considerable selection effects atHbI, and stress the unreliability of allele frequencies at this locus for use in studies of the genetic population structure of cod.Contribution No. 229, Biological Station, N-7000 Trondheim, Norway     

Digestibility of chitin in cod,Gadus morhua,in vivo

Sixteen cod,Gadus morhua (L.), were individually fed a single ration of shrimps,Crangon allmanni. Four fish were killed and examined 6, 12, 24 and 48 h after the fish had been fed. Chitinase activities were measured in the extracts of stomach contents, stomach tissue, pyloric caecae, intestinal contents and intestinal tissue. The level of enzyme activity in different parts of the digestive tract was shown to be dependent on the phase of the digestive process. High concentrations of the chitin degradation product N-acetyl-D-glucosamine were determined in the stomach and in the intestinal contents. Based on the chitin concentration in the food organisms and the individual food uptake, the amount of chitin consumed by each fish could be calculated. Only up to 9% of the ingested chitin was recovered from the intestinal contents of the fish at any given time after feeding (6, 12, 24 and 48 h). In addition, only 2.4% of the chitin consumed with the food could be recovered in the collected faeces of the fish. The 4 cod killed 48 h after feeding had completely emptied their stomach. Chitin digestion in these fish was calculated to have been 90%.     

Homing and group cohesion in Atlantic cod Gadus morhua revealed by tagging experiments

Homing behaviour and group cohesion in Atlantic cod Gadus morhua from the northern Gulf of St Lawrence were studied based on tagging-recapture data from two periods, the 1980s and a recent period from 1996 to 2008. Two or more tags from a single tagging experiment were frequently recovered together in subsequent years. The null hypothesis was tested that the frequency of matching tag recoveries occurred by chance only through random mixing of tagged G. morhua before their recapture by the commercial fishery. The alternative hypothesis was that non-random, positive association (group cohesion) existed among tagged individuals that persisted through time and during migrations. Results show that the G. morhua population exhibits a homing behaviour, with temporal stability across seasons and years: 50% of recaptured fish in the recent period were caught <34 km from their mark site, even 3 years after release. In the 1980s, G. morhua were located at <10 km from their release site 1 year after tagging during summer and at <16 km during spring and autumn combined. Despite the increasing distance between the mark and recapture sites over time, the difference was not significant. In addition, occurrences of two or more tagged fish from the same release event that were caught together indicated a non-random association among individual fish for periods of one to several years and through migrations over several hundred kilometres. Hence G. morhua showed group cohesion in addition to site fidelity. These two interacting behaviours may be fundamental for the rebuilding and conservation of depleted fish stocks.     

Feed intake and tissue distribution of florfenicol in cod (Gadus morhua) administered feed with different fat contents

This study was performed to examine the appetite and the corresponding plasma and tissue distribution of florfenicol when administered to healthy groups of cod using medicated and non‐medicated salmonid and marine feeds. Marine feed contains approximately 18% fat whereas salmonid feed contains approx. 30% fat. Two groups of fish were medicated with florfenicol at a dosage of 10 mg kg^?1 day^?1 for 10 consecutive days when the drug was administered either via marine or salmonid pellets. Two groups of fish also received either non‐medicated marine or salmonid pellets. Twenty‐four hours after giving the medicated marine feed, 14 out of 20 fish contained detectable concentrations of florfenicol with mean values (n = 14) of 4.67 ± 4.02 μg ml^?1 in plasma, 2.29 ± 2.11 μg g^?1 in muscle and 0.79 ± 0.69 μg g^?1 in the liver. In the fish given medicated salmonid feed, 18 of 20 fish contained detectable concentrations of florfenicol with mean values (n = 18) of 1.77 ± 1.84 μg ml^?1 in plasma, 0.75 ± 0.66 μg g^?1 in muscle and 0.30 ± 0.25 μg g^?1 in the liver. Decreased feed intake of the salmonid feed, both medicated and non‐medicated, was noted when compared to medicated and non‐medicated marine feed. No difference in feed consumption was registered between medicated and non‐medicated marine feed, however a difference was noted between the medicated and non‐medicated salmonid feed.