Characterization of nutrients and contaminants in fish sludge from Atlantic salmon (Salmo salar L.) production sites - A future resource.

A total of 47 fish sludge samples from commercial land-based Atlantic salmon (Salmo salar) farms in Norway were assessed for their nutrient composition, presence of various legacy contaminants and a wide spectrum of contaminants of emerging concern, veterinary medicines as well as selected salmonid pathogenic bacteria and virus. The aim was to document the levels of desirable and undesirable components in fish sludge in relation to a potential future use of sludge as invertebrate feed. The samples had variable, but relatively high protein and fat contents, indicating a high load of undigested feed in some of the sludge samples. Fatty acid analysis showed the presence of essential omega-3 fatty acids. In terms of undesirable substances, 43% and 84% of the sludge samples contained levels of arsenic and cadmium, respectively, which exceeded the EU Maximum Levels established for complete animal feed. The concentrations of copper, zinc, iron and aluminum were highly variable in the sludge samples. The concentrations of dioxins, sum PCB6, and chlorinated pesticides were all below the Maximum Levels for animal feed. Of the 18 per- and polyfluoroalkyl substances (PFAS) only one compound (L-PFOS) was present at measurable levels. None of the samples had detectable levels of veterinary medicines, salmonid virus or bacteria. Performing a suspect and non-target screening of the sludge samples identified 18 compounds, including four pharmaceuticals, plastic-related products and the UV filter benzophenone, warranting further investigations. Overall, the results from this study show that fish sludge is a nutrient-rich resource; however, undesirable substances, originating from the feed or from treatment of sludge may be present.

Draft genome sequences of 13 Tenacibaculum maritimum isolates from farmed Norwegian Cyclopterus lumpus (lumpfish) and Scophthalmus maximus (turbot).

Thirteen bacterial isolates of Tenacibaculum maritimum were sequenced and assembled. The strains were isolated from four disease outbreaks in farmed marine fish in Norway. Eight isolates were from Cyclopterus lumpus (lumpfish), and five were from Scophthalmus maximus (turbot). Overall, sequence similarity did not correlate with host species or geographic location.

Phylogeography and host specificity of Pasteurellaceae pathogenic to sea-farmed fish in the north-east Atlantic.

The present study was undertaken to address the recent spate of pasteurellosis outbreaks among sea-farmed Atlantic salmon (Salmo salar) in Norway and Scotland, coinciding with sporadic disease episodes in lumpfish (Cyclopterus lumpus) used for delousing purposes in salmon farms. Genome assemblies from 86 bacterial isolates cultured from diseased salmon or lumpfish confirmed them all as bona fide members of the Pasteurellaceae family, with phylogenetic reconstruction dividing them into two distinct branches sharing <88% average nucleotide identity. These branches therefore constitute two separate species, namely Pasteurella skyensis and the as-yet invalidly named "Pasteurella atlantica". Both species further stratify into multiple discrete genomovars (gv.) and/or lineages, each being nearly or fully exclusive to a particular host, geographic region, and/or time period. Pasteurellosis in lumpfish is, irrespective of spatiotemporal origin, linked almost exclusively to the highly conserved "P. atlantica gv. cyclopteri" (Pac). In contrast, pasteurellosis in Norwegian sea-farmed salmon, dominated since the late-1980s by "P. atlantica gv. salmonicida" (Pas), first saw three specific lineages (Pas-1, -2, and -3) causing separate, geographically restricted, and short-lived outbreaks, before a fourth (Pas-4) emerged recently and became more widely disseminated. A similar situation involving P. skyensis (Ps) has apparently been unfolding in Scottish salmon farming since the mid-1990s, where two historic (Ps-1 and -2) and one contemporary (Ps-3) lineages have been recorded. While the epidemiology underlying all these outbreaks/epizootics remains unclear, repeated detection of 16S rRNA gene amplicons very closely related to P. skyensis and "P. atlantica" from at least five cetacean species worldwide raises the question as to whether marine mammals may play a part, possibly as reservoirs. In fact, the close relationship between the studied isolates and Phocoenobacter uteri associated with harbor porpoise (Phocoena phocoena), and their relatively distant relationship with other members of the genus Pasteurella, suggests that both P. skyensis and "P. atlantica" should be moved to the genus Phocoenobacter.

MLVA genotyping of Moritella viscosa reveals serial emergence of novel, host-specific clonal complexes in Norwegian salmon farming.

A Multi-Locus Variable number of tandem repeat Analysis (MLVA) genotyping scheme was developed for the epidemiological study of Moritella viscosa, which causes 'winter ulcer' predominantly in sea-reared Atlantic salmon (Salmo salar L.). The assay involves multiplex PCR amplification of six Variable Number of Tandem Repeat (VNTR) loci, followed by capillary electrophoresis and data interpretation. A collection of 747 spatiotemporally diverse M. viscosa isolates from nine fish species was analysed, the majority from farmed Norwegian salmon. MLVA distributed 76% of the isolates across three major clonal complexes (CC1, CC2 and CC3), with the remaining forming minor clusters and singletons. While 90% of the salmon isolates belong to either CC1, CC2 or CC3, only 20% of the isolates recovered from other fish species do so, indicating a considerable degree of host specificity. We further highlight a series of 'clonal shifts' amongst Norwegian salmon isolates over the 35-year sampling period, with CC1 showing exclusive predominance prior to the emergence of CC2, which was later supplanted by CC3, before the recent re-emergence of CC1. Apparently, these shifts have rapidly swept the entire Norwegian coastline and conceivably, as suggested by typing of a small number of non-Norwegian isolates, the Northeast Atlantic region as a whole.

Draft Genome Sequences of 23 Tenacibaculum Isolates from Farmed Norwegian Lumpfish, Cyclopterus lumpus.

Draft genome sequences of 23 Tenacibaculum sp. strains that were isolated from Cyclopterus lumpus (lumpfish) were investigated to elucidate possible routes of transmission between Salmo salar (Atlantic salmon) and lumpfish.

Tenacibaculosis in Norwegian Atlantic salmon (Salmo salar) cage-farmed in cold sea water is primarily associated with Tenacibaculum finnmarkense genomovar finnmarkense.

Skin conditions associated with Tenacibaculum spp. constitute a significant threat to the health and welfare of sea-farmed Atlantic salmon (Salmo salar L.) in Norway. Fifteen presumptive tenacibaculosis outbreaks distributed along the Norwegian coast during the late winter and spring of 2018 were investigated. Bacteriological culture confirmed the presence of Tenacibaculum spp. Seventy-six isolates cultured from individual fish were selected and subjected to whole-genome sequencing and MALDI-TOF MS analysis. Average nucleotide identity and MALDI-TOF analyses confirmed the presence of T. finnmarkense and T. dicentrarchi, with further division of T. finnmarkense into genomovars (gv.) finnmarkense and ulcerans. Core genome multilocus sequence typing (cgMLST) and single-nucleotide polymorphism (SNP) analyses identified the presence of a genetically conserved cluster of gv. finnmarkense isolates against a background of relatively genetically diverse gv. finnmarkense and gv. ulcerans isolates in 13 of the 15 studied cases. This clustering strongly suggests a link between T. finnmarkense gv. finnmarkense and development of clinical tenacibaculosis in sea-farmed Norwegian salmon in the late winter and spring. Analysis of 25 Tenacibaculum isolates collected during the spring of 2019 from similar cases identified a similar distribution of genotypes. Low water temperatures were common to all cases, and most incidences involved relatively small fish shortly after sea transfer, suggesting that these fish are particularly predisposed to Tenacibaculum infection.

Multilocus sequence analysis reveals different lineages of Pseudomonas anguilliseptica associated with disease in farmed lumpfish (Cyclopterus lumpus L.).

The bacterium Pseudomonas anguilliseptica has in recent years emerged as a serious threat to production of lumpfish in Norway. Little is known about the population structure of this bacterium despite its association with disease in a wide range of different fish species throughout the world. The phylogenetic relationships between 53 isolates, primarily derived from diseased lumpfish, but including a number of reference strains from diverse geographical origins and fish species, were reconstructed by Multi-Locus Sequence Analysis (MLSA) using nine housekeeping genes (rpoB, atpD, gyrB, rpoD, ileS, aroE, carA, glnS and recA). MLSA revealed a high degree of relatedness between the studied isolates, altough the seven genotypes identified formed three main phylogenetic lineages. While four genotypes were identified amongst Norwegian lumpfish isolates, a single genotype dominated, irrespective of geographic origin. This suggests the existence of a dominant genotype associated with disease in production of lumpfish in Norwegian aquaculture. Elucidation of the population structure of the bacterium has provided valuable information for potential future vaccine development.

Early detection of salmonid alphavirus in seawater from marine farm sites of Atlantic salmon Salmo salar.

The traditional strategy for national surveillance of salmonid alphavirus (SAV) infection in Norwegian fish farms relies on a costly, time-consuming, and resource-demanding approach based on the monthly sampling of fish from all marine farms with salmonids. In order to develop an alternative surveillance method, a water filtration method was tested in parallel with the ongoing surveillance program at 7 Norwegian marine farm sites of Atlantic salmon Salmo salar L. with no current suspicion of SAV infection. During the period from May 2019 to January 2020, seawater samples were collected from the top layer water inside all net-pens at these 7 sites. The samples were concentrated for SAV by filtration through an MF-Millipore™ electronegative membrane filter, followed by rinsing with NucliSENS® Lysis Buffer, before RNA extraction and analysis by RT-qPCR. SAV was detected from seawater at an earlier stage compared to traditional sampling methods, at all sites where the fish tested positive for SAV. A significant negative relationship was observed at all sites between the SAV concentration found in seawater samples and the number of days until SAV was detected in the fish. This means that the fewer the SAV particles in the seawater, the more days it took until SAV was detected in the fish samples. Based on this, sampling of seawater every month for the surveillance of SAV has a great potential as an alternative method for early detection of SAV in Atlantic salmon farms.

Use of anticholinergic drugs in older patients. / Bruk av legemidler med antikolinerg virkning hos eldre.

Many medicines prescribed for older patients have an unintended anticholinergic effect in addition to the primary intended effect. This may lead to adverse reactions such as dizziness and memory loss, particularly in older people. Before prescribing new medicines, it is therefore important to assess the total anticholinergic burden by means of specific screening tools, such as AGS Beers Criteria® and the STOPP/START criteria. If the anticholinergic burden is high, drugs should be replaced or discontinued.

Tenacibaculum piscium sp. nov., isolated from skin ulcers of sea-farmed fish, and description of Tenacibaculum finnmarkense sp. nov. with subdivision into genomovars finnmarkense and ulcerans.

Results of previous multilocus sequence and whole-genome-based analyses have suggested that a homogeneous group of isolates belonging to the genus Tenacibaculum, represented by strain TNO020T and associated with skin ulcer development in sea-farmed fish, represents an as-yet-undescribed species. Comparative whole-genome analysis performed in the present study clustered five isolates, including TNO020T, in a distinct lineage within the genus Tenacibaculum. Phenotypic differences, high intra-cluster average nucleotide identity (ANI) values and low ANI values with other Tenacibaculum species support the proposal of a novel species, for which we propose the name Tenacibaculum piscium sp. nov. with strain TNO020T (=CCUG 73833T=NCIMB 15240T) as the type strain. Further, large-scale genome analyses confirmed the existence of two different phylogenetic lineages within 'T. finnmarkense', a species effectively but not validly published previously. ANI values just above the species delineation threshold of 95-96 % confirmed that both lineages belong to the same species. This result was also supported by DNA-DNA hybridization values. Phenotypically, the two conspecific lineages are distinguishable by differences in growth temperature range and ability to degrade l-proline. For the group of isolates already commonly known as 'T. finnmarkense', we propose the name Tenacibaculum finnmarkense sp. nov., with strain TNO006T (=CCUG 73831T=NCIMB 15238T) as the type strain. We further propose the subdivision of T. finnmarkense sp. nov. into two genomovars, T. finnmarkense genomovar finnmarkense with strain TNO006T (=CCUG 73831T=NCIMB 15238T) as the type strain and T. finnmarkense genomovar ulcerans with strain TNO010T (=CCUG 73832T=NCIMB 15239T) as the type strain.

Anticoagulant-associated adverse drug reactions in 2013-15. / Bivirkninger ved bruk av antikoagulasjonsmidler i 2013­15.

BACKGROUND: The aim of this study was to obtain a better insight into the adverse effects profiles of the new direct-acting oral anticoagulants (DOACs). MATERIAL AND METHOD: A review was undertaken of all reports of adverse effects for warfarin, dabigatran, rivaroxaban and apixaban reported to the regional medicines information and pharmacovigilance centres (RELIS) in the period June 2013-May 2015. RESULTS: Approximately 65 000 persons used direct-acting oral anticoagulants and 80 000 used warfarin in the period of the study. A total of 409 reports of adverse effects were included. Altogether 55 % of the reports applied to men. In 76 % of the reports for direct-acting oral anticoagulants and 85 % for warfarin, the patients were more than 70 years of age. The most common adverse effects were haemorrhages (48 % for direct-acting oral anticoagulants and 75 % for warfarin), most of which were cerebral haemorrhages (91 for direct-acting oral anticoagulants and 92 for warfarin). Blood clots (therapeutic failure), cognitive effects, headache and hair loss were some of the other adverse effects. The highest comorbidity was among the patients who died. The number of reported deaths was highest for rivaroxaban (1.1 deaths/1000 users) with a declining incidence for apixaban (0.9 ‰), dabigatran (0.7 ‰) and warfarin (0.6 ‰). There were different degrees of reporting for these medications, and the spontaneous reporting system cannot therefore be used to compare the incidence of adverse effects for the drugs. INTERPRETATION: Adverse effects, including serious effects, may occur when using all anticoagulants. Factors that may increase the risk of adverse effects are advanced age, high comorbidity, reduced renal function, and polypharmacy.

Genomic Diversity and Evolution of the Fish Pathogen Flavobacterium psychrophilum.

Flavobacterium psychrophilum, the etiological agent of rainbow trout fry syndrome and bacterial cold-water disease in salmonid fish, is currently one of the main bacterial pathogens hampering the productivity of salmonid farming worldwide. In this study, the genomic diversity of the F. psychrophilum species is analyzed using a set of 41 genomes, including 30 newly sequenced isolates. These were selected on the basis of available MLST data with the two-fold objective of maximizing the coverage of the species diversity and of allowing a focus on the main clonal complex (CC-ST10) infecting farmed rainbow trout (Oncorhynchus mykiss) worldwide. The results reveal a bacterial species harboring a limited genomic diversity both in terms of nucleotide diversity, with ~0.3% nucleotide divergence inside CDSs in pairwise genome comparisons, and in terms of gene repertoire, with the core genome accounting for ~80% of the genes in each genome. The pan-genome seems nevertheless "open" according to the scaling exponent of a power-law fitted on the rate of new gene discovery when genomes are added one-by-one. Recombination is a key component of the evolutionary process of the species as seen in the high level of apparent homoplasy in the core genome. Using a Hidden Markov Model to delineate recombination tracts in pairs of closely related genomes, the average recombination tract length was estimated to ~4.0 Kbp and the typical ratio of the contributions of recombination and mutations to nucleotide-level differentiation (r/m) was estimated to ~13. Within CC-ST10, evolutionary distances computed on non-recombined regions and comparisons between 22 isolates sampled up to 27 years apart suggest a most recent common ancestor in the second half of the nineteenth century in North America with subsequent diversification and transmission of this clonal complex coinciding with the worldwide expansion of rainbow trout farming. With the goal to promote the development of tools for the genetic manipulation of F. psychrophilum, a particular attention was also paid to plasmids. Their extraction and sequencing to completion revealed plasmid diversity that remained hidden to classical plasmid profiling due to size similarities.

Comparative Genomics of Tenacibaculum dicentrarchi and "Tenacibaculum finnmarkense" Highlights Intricate Evolution of Fish-Pathogenic Species.

The genus Tenacibaculum encompasses several species pathogenic for marine fish. Tenacibaculum dicentrarchi and "Tenacibaculum finnmarkense" (Quotation marks denote species that have not been validly named.) were retrieved from skin lesions of farmed fish such as European sea bass or Atlantic salmon. They cause a condition referred to as tenacibaculosis and severe outbreaks and important fish losses have been reported in Spanish, Norwegian, and Chilean marine farms. We report here the draft genomes of the T. dicentrarchi and "T. finnmarkense" type strains. These genomes were compared with draft genomes from field isolates retrieved from Chile and Norway and with previously published Tenacibaculum genomes. We used Average Nucleotide Identity and core genome-based phylogeny as a proxy index for species boundary delineation. This work highlights evolution of closely related fish-pathogenic species and suggests that homologous recombination likely contributes to genome evolution. It also corrects the species affiliation of strain AYD7486TD claimed by Grothusen et al. (2016).

Multilocus sequence analysis reveals extensive genetic variety within Tenacibaculum spp. associated with ulcers in sea-farmed fish in Norway.

Skin ulcer development in sea-reared salmonids, commonly associated with Tenacibaculum spp., is a significant fish welfare- and economical problem in Norwegian aquaculture. A collection of 89 Tenacibaculum isolates was subjected to multilocus sequence analysis (MLSA). The isolates were retrieved from outbreaks of clinical disease in farms spread along the Norwegian coast line from seven different fish species over a period of 19 years. MLSA analysis reveals considerable genetic diversity, but allows identification of four main clades. One clade encompasses isolates belonging to the species T. dicentrarchi, whereas three clades encompass bacteria that likely represent novel, as yet undescribed species. The study identified T. maritimum in lumpsucker, T. ovolyticum in halibut, and has extended the host and geographic range for T. soleae, isolated from wrasse. The overall lack of clonality and host specificity, with some indication of geographical range restriction argue for local epidemics involving multiple strains. The diversity of Tenacibaculum isolates from fish displaying ulcerative disease may complicate vaccine development.

[Drug safety associated with the change of digitalis drug in Norway]. / Legemiddelsikkerhet ved bytte av digitalispreparat i Norge.

BACKGROUND In 2011, following a period with delivery problems, the only registered digitoxin drug in Norway was replaced with digoxin. As a result, approximately 21 000 patients had to replace digitoxin with digoxin. There are important pharmacokinetic differences between digitoxin and digoxin (the general term for both drugs is digitalis), which must be taken into account when changing therapy. The aim of this study was to investigate compliance of drug security, during the transition from digitoxin to digoxin in Norway.MATERIAL AND METHOD Enquiries addressed to the Norwegian Poison Information Centre and reports of fatal adverse effects to the Regional Drug Information Centres (RELIS) regarding intake of digitalis were analysed. Serum concentrations of digitoxin and digoxin analyzed at Oslo University Hospital were reviewed. All data sources were reviewed for the years 2010-2014 and patients > 20 years were included.RESULTS The total number of enquiries addressed to the Norwegian Poison Information Centre, fatal adverse drug reactions reported to RELIS, and patient samples in the toxic range analyzed at Oslo University Hospital increased from 2012, timewise related to the transition to digoxin.INTERPRETATION Despite extensive information from the Norwegian Medicines Agency, a small, transient increase was observed in the number of overdoses and reported deaths from digitalis related to change in therapy. The cause of the overdose was in many cases unknown. This study revealed several cases of incorrect dosage, simultaneous use of digitoxin and digoxin, and washout time that was insufficient or lacking before initiation of digoxin.

Multilocus sequence analysis of the marine bacterial genus Tenacibaculum suggests parallel evolution of fish pathogenicity and endemic colonization of aquaculture systems.

The genus Tenacibaculum, a member of the family Flavobacteriaceae, is an abundant component of marine bacterial ecosystems that also hosts several fish pathogens, some of which are of serious concern for marine aquaculture. Here, we applied multilocus sequence analysis (MLSA) to 114 representatives of most known species in the genus and of the worldwide diversity of the major fish pathogen Tenacibaculum maritimum. Recombination hampers precise phylogenetic reconstruction, but the data indicate intertwined environmental and pathogenic lineages, which suggests that pathogenicity evolved independently in several species. At lower phylogenetic levels recombination is also important, and the species T. maritimum constitutes a cohesive group of isolates. Importantly, the data reveal no trace of long-distance dissemination that could be linked to international fish movements. Instead, the high number of distinct genotypes suggests an endemic distribution of strains. The MLSA scheme and the data described in this study will help in monitoring Tenacibaculum infections in marine aquaculture; we show, for instance, that isolates from tenacibaculosis outbreaks in Norwegian salmon farms are related to T. dicentrarchi, a recently described species.

Multilocus sequence typing identifies epidemic clones of Flavobacterium psychrophilum in Nordic countries.

Flavobacterium psychrophilum is the causative agent of bacterial cold water disease (BCWD), which affects a variety of freshwater-reared salmonid species. A large-scale study was performed to investigate the genetic diversity of F. psychrophilum in the four Nordic countries: Denmark, Finland, Norway, and Sweden. Multilocus sequence typing of 560 geographically and temporally disparate F. psychrophilum isolates collected from various sources between 1983 and 2012 revealed 81 different sequence types (STs) belonging to 12 clonal complexes (CCs) and 30 singleton STs. The largest CC, CC-ST10, which represented almost exclusively isolates from rainbow trout and included the most predominant genotype, ST2, comprised 65% of all isolates examined. In Norway, with a shorter history (<10 years) of BCWD in rainbow trout, ST2 was the only isolated CC-ST10 genotype, suggesting a recent introduction of an epidemic clone. The study identified five additional CCs shared between countries and five country-specific CCs, some with apparent host specificity. Almost 80% of the singleton STs were isolated from non-rainbow trout species or the environment. The present study reveals a simultaneous presence of genetically distinct CCs in the Nordic countries and points out specific F. psychrophilum STs posing a threat to the salmonid production. The study provides a significant contribution toward mapping the genetic diversity of F. psychrophilum globally and support for the existence of an epidemic population structure where recombination is a significant driver in F. psychrophilum evolution. Evidence indicating dissemination of a putatively virulent clonal complex (CC-ST10) with commercial movement of fish or fish products is strengthened.

DNA gyrase and topoisomerase IV mutations in quinolone-resistant Flavobacterium psychrophilum isolated from diseased salmonids in Norway.

Flavobacterium psychrophilum is the causative agent of the recognized diseases 'bacterial coldwater disease' and 'rainbow trout fry syndrome' and is found in many farmed freshwater and marine fish species. In Norway, the bacterium has mainly been isolated from Atlantic salmon (Salmo salar L.) and brown trout (Salmo trutta L.). In the present study, 26 isolates from Norwegian farmed salmonids were examined. All isolates were tested for susceptibility towards various antibacterial drugs by the disk diffusion method, and minimum inhibitory concentration values for oxolinic acid and flumequine were established for selected isolates. All isolates from rainbow trout displayed reduced susceptibility towards quinolones, while brown trout and Atlantic salmon isolates were susceptible. The quinolone resistance determining regions (QRDRs) of the gyrA, gyrB, parC, and parE genes were sequenced. Sequence analysis of the QRDR of gyrA in quinolone resistant isolates revealed a threonine:arginine amino acid substitution at position 82 in all 16 isolates from Norwegian rainbow trout and a single reference strain isolated from rainbow trout in Sweden. No evidence for plasmid-mediated quinolone resistance was found in any of the isolates. Pulsed-field gel electrophoresis and phylogenetic analysis of parC and gyrB sequences indicate a clonal relationship between rainbow trout isolates.