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.     

Changes in arterial PO₂, physiological blood parameters and intracellular antioxidants in free-swimming Atlantic cod (Gadus morhua) exposed to varying levels of hyperoxia

Free-swimming Atlantic cod (Gadus morhua) were exposed to water oxygen pressures (P _wO₂) ranging from 18.1 to 41.5 kPa and sampled for blood using an indwelling caudal artery cannula. Arterial blood oxygen pressure (P _aO₂) increased with increasing P _wO₂, from 12.0 kPa in normoxia (18.1 kPa) to 34.2 kPa in the highest hyperoxic level tested (41.5 kPa). Blood CO₂ pressure and plasma bicarbonate concentration increased with P _wO₂, indicating reduced ventilation with increased P _wO₂. Plasma glucose, sodium and potassium were not affected by water oxygen level. Blood oxidative stress biomarkers, reduced glutathione, oxidized glutathione and the oxidative stress index (ratio between oxidized and total glutathione) differed intermittently between normoxia and hyperoxia. The oxidative stress index was higher in the blood of exposed compared to unexposed control cod. Together with elevated P _aO₂, these findings suggest increased production of reactive oxygen species and increased oxidative stress in Atlantic cod exposed to hyperoxia.     

Diversity of Flavobacterium psychrophilum and the potential use of its phages for protection against bacterial cold water disease in salmonids

Flavobacterium psychrophilum causes rainbow trout fry syndrome (RTFS) and cold water disease (CWD) in salmonid aquaculture. We report characterization of F. psychrophilum strains and their bacteriophages isolated in Chilean salmonid aquaculture. Results suggest that under laboratory conditions phages can decrease mortality of salmonids from infection by their F. psychrophilum host strain. Twelve F. psychrophilum isolates were characterized, with DNA restriction patterns showing low diversity between strains despite their being obtained from different salmonid production sites and from different tissues. We isolated 15 bacteriophages able to infect some of the F. psychrophilum isolates and characterized six of them in detail. DNA genome sizes were close to 50 Kbp and corresponded to the Siphoviridae and Podoviridae families. One isolate, 6H, probably contains lipids as an essential virion component, based on its chloroform sensitivity and low buoyant density in CsCl. Each phage isolate rarely infected F. psychrophilum strains other than the strain used for its enrichment and isolation. Some bacteriophages could decrease mortality from intraperitoneal injection of its host strain when added together with the bacteria in a ratio of 10 plaque-forming units per colony-forming unit. While we recognize the artificial laboratory conditions used for these protection assays, this work is the first to demonstrate that phages might be able protect salmonids from RTFS or CWD.     

Linking watershed and microhabitat characteristics: effects on production of Atlantic salmonids (Salmo salar and Salmo trutta)

The spatial scale of environmental factors influencing population dynamics ranges from microhabitat to continental or even global scales. Integration of multiple spatial scales is important in order to understand links between environmental variation and population processes. In the present study, we investigate how multiscale drivers influence the production of stream‐rearing Atlantic salmonids (Atlantic salmon, Salmo salar L. and brown trout, Salmo trutta L.) measured in terms of abundance. Variation in juvenile production was studied using data from single‐pass electrofishing surveys (measured as biomass per m²) from nine rivers. These data were combined with habitat data ranging from an important in‐stream microhabitat variable (shelter availability) to properties of the catchment. Variation in productivity within and among rivers was affected by both properties of in‐stream habitat and catchment properties. Shelter availability and the proportion of the catchment consisting of cultivated land and lakes influenced biomass positively, while catchment area had the opposite effect. For a different set of rivers (N = 20), river gradient and catchment area were shown to positively affect the amount of shelter. Finally, the variables identified in the two preceding analysis were included in the analysis of population productivity using catch statistics from 160 rivers. The proportion of cultivated land and lakes, estimated shelter availability were found to have positive effects. In addition, temperature had a positive effect, while river width had a negative effect. This study shows that combining multiple‐scale environmental factors can explain a substantial proportion of variation in population productivity among and within the populations of Atlantic salmonids.     

Infection routes of Aeromonas salmonicida in rainbow trout monitored in vivo by real‐time bioluminescence imaging

Recent development of imaging tools has facilitated studies of pathogen infections in vivo in real time. This trend can be exemplified by advances in bioluminescence imaging (BLI), an approach that helps to visualize dissemination of pathogens within the same animal over several time points. Here, we employ bacterial BLI for examining routes of entry and spread of Aeromonas salmonicida susbp. salmonicida in rainbow trout. A virulent Danish A. salmonicida strain was tagged with pAKgfplux1, a dual‐labelled plasmid vector containing the mutated gfpmut3a gene from Aequorea victoria and the luxCDABE genes from the bacterium Photorhabdus luminescens. The resulting A. salmonicida transformant exhibited growth properties and virulence identical to the wild‐type A. salmonicida, which made it suitable for an experimental infection, mimicking natural conditions. Fish were infected with pAKgfplux1 tagged A. salmonicida via immersion bath. Colonization and subsequent tissue dissemination was followed over a 24‐h period using the IVIS spectrum imaging workstation. Results suggest the pathogen's colonization sites are the dorsal and pectoral fin and the gills, followed by a progression through the internal organs and an ensuing exit via the anal opening. This study provides a tool for visualizing colonization of A. salmonicida and other bacterial pathogens in fish.     

Detection and quantification of Aeromonas salmonicida in fish tissue by real‐time PCR

Furunculosis, a septicaemic infection caused by the bacterium Aeromonas salmonicida subsp. salmonicida, currently causes problems in Danish seawater rainbow trout production. Detection has mainly been achieved by bacterial culture, but more rapid and sensitive methods are needed. A previously developed real‐time PCR assay targeting the plasmid encoded aopP gene of A. salmonicida was, in parallel with culturing, used for the examination of five organs of 40 fish from Danish freshwater and seawater farms. Real‐time PCR showed overall a higher frequency of positives than culturing (65% of positive fish by real‐time PCR compared to 30% by a culture approach). Also, no real‐time PCR‐negative samples were found positive by culturing. A. salmonicida was detected by real‐time PCR, though not by culturing, in freshwater fish showing no signs of furunculosis, indicating possible presence of carrier fish. In seawater fish examined after an outbreak and antibiotics treatment, real‐time PCR showed the presence of the bacterium in all examined organs (1–482 genomic units mg^?1). With a limit of detection of 40 target copies (1–2 genomic units) per reaction, a high reproducibility and an excellent efficiency, the present real‐time PCR assay provides a sensitive tool for the detection of A. salmonicida.     

Local adaptation studies and conservation: Parasite–host interactions between the endangered freshwater mussel Unio crassus and its host fish

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Effect of supplemented fungal phytase on performance and phosphorus availability by phosphorus-depleted juvenile rainbow trout (Oncorhynchus mykiss), and on the magnitude and composition of phosphorus waste output

The effect of a supplemental fungal phytase on performance and phosphorus availability by juvenile rainbow trout fed diets with a high inclusion of plant based protein and on the magnitude and composition of the waste phosphorus production was tested in a 2 × 3 factorial design at a temperature of 11 °C. Two factors comprised of two dietary fungal phytase levels (0 or 1400 U kg^? 1 feed^? 1), and three dietary total phosphorus levels (0.89, 0.97 or 1.12%). All fish were acclimated to the lowest total phosphorus diet for 16 days, which included 0.29% phytate-phosphorus and no supplemental fungal phytase, to ensure that they were depleted of phosphorus prior to the feeding trial.Growth and feed conversion ratios were not significantly affected by the increasing dietary phosphorus level or supplemental fungal phytase. Phosphorus availability increased significantly as a result of phytase supplementation, reaching an upper level of 74% at an available dietary phosphorus concentration of 0.71%. Adding fungal phytase to the diets improved the availability of phytate-phosphorus from an average of 6 to 64%. The fish retained 53–79% of the ingested phosphorus, while 24–44% was recovered in the faeces. The particulate phosphorus waste output was significantly higher in faeces from fish fed diets without fungal phytase compared to fish fed phytase supplemented diets. The dissolved/suspended phosphorus waste output represented 2–13% of the ingested phosphorus, and there was a significant increase in the dissolved/suspended phosphorus waste output from fish fed the phytase supplemented diet containing 0.71% available phosphorus, suggesting that the phosphorus requirement was reached at this phosphorus level. Consistent with this, there was a substantial increase in the dissolved/suspended phosphorus waste output from fish fed the phytase supplemented diet containing 0.81% available phosphorus, suggesting that the phosphorus requirement was exceeded in this group.This study demonstrated that phytase supplementation will be advantageous to the fish and the environment if supplemented to low-phosphorus diets containing a large share of plant-derived protein. Conversely, the results demonstrated that fungal phytase should not be supplemented to diets in which the available phosphorus level already meets the requirement of the fish, as this will lead to a significant increase in the dissolved/suspended phosphorus waste output.     

Growth performance,nutrient digestibility and intestinal morphology of rainbow trout (Oncorhynchus mykiss) fed graded levels of the yeasts Saccharomyces cerevisiae and Wickerhamomyces anomalus

In a 10‐week study, we evaluated the effects of replacing 20%, 40% or 60% of fishmeal (present in control diet at 300 g/kg) on a digestible protein basis with yeast Saccharomyces cerevisiae or a yeast mixture of Wickerhamomyces anomalus and S. cerevisiae on growth performance, nutrient digestibility, nutrient retention and intestinal health of rainbow trout (Oncorhynchus mykiss). Triplicate tanks with 35 rainbow trout (144.7 ± 25.1 g mean ± SEM) were fed rations of 1.5% of total biomass per tank. Replacement of 60% of fishmeal with yeast mixture resulted in lower specific growth rate of 1.0% versus 1.2%/day for other diets. Apparent digestibility coefficients for crude protein and most amino acids were highest in fish fed fishmeal‐based diet, with similar values for fish fed the diet with 20% replacement with yeast mixture. Diet with 20% replacement with yeast mixture resulted in highest phosphorus digestibility. Replacement of 60% of fishmeal with S. cerevisiae resulted in oedematous mucosal fold tips in the proximal intestine. The results of this study suggest that these yeasts can replace up to 40% of fishmeal under current inclusion levels in diets for rainbow trout without compromising growth performance, nutrient digestibility or intestinal health.     

A test of the individual feeding activity and food size preference in rainbow trout using demand feeders

Feeding activity and food size preferences were investigated in three groups of rainbow trout with initial weights ranging from 200 to 1000 g. Fish were given the option of releasing food from either of two demand feeding devices that contained either 6 mm or 9 mm food pellets. Individual biting activity (to activate release of food) was continuously monitored using specially designed passive integrated transponders (PIT-tags). In each of the groups, biting activity was strongly influenced by three individuals, which accounted for 70–85% of the total. Thus, high bite activity by few individuals indicates the development of social dominance hierarchies. There was no statistically significant correlation between number of bites and growth rate, indicating that the fish that operated the demand feeders did not gain any growth advantage. Trout that dominated the actuation of the feeders were found among both medium- and large-sized fish. There was no evidence of active food size selection, and the fish that most frequently operated the feeders defended one area of the tank rather than preferring the trigger that would have delivered food of the optimal size.