Publications by authors named "Inger Dalsgaard"

51 Publications

Genome-informed approach to identify genetic determinants of Flavobacterium psychrophilum phage susceptibility.

Environ Microbiol 2021 May 14. Epub 2021 May 14.

Marine Biological Section, Department of Biology, University of Copenhagen, Helsingør, Denmark.

The fish pathogen Flavobacterium psychrophilum infects farmed salmonids worldwide, and application of bacteriophages has been suggested for controlling disease outbreaks in aquaculture. Successful application of phages requires detailed knowledge about the variability in phage susceptibility of the host communities. In this study, we analysed the genetic diversity of F. psychrophilum hosts and phages from the Baltic Sea area to identify genetic determinants of phage-host interaction patterns. A host range analysis of 103 phages tested against 177 F. psychrophilum strains (18 231 phage-host interactions) identified nine phage clusters, infecting from 10% to 91% of the strain collection. The core genome-based comparison of 35 F. psychrophilum isolates revealed an extremely low overall genomic diversity (>99.5% similarity). However, a small subset of 16 ORFs, including genes involved in the type IX secretion system (T9SS), gliding motility and hypothetical cell-surface related proteins, exhibited a highly elevated genetic diversity. These specific genetic variations were linked to variability in phage infection patterns obtained from experimental studies, indicating that these genes are key determinants of phage susceptibility. These findings provide novel insights on the molecular mechanisms determining phage susceptibility in F. psychrophilum and emphasizes the importance of phages as drivers of core genomic diversity in this pathogen.
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http://dx.doi.org/10.1111/1462-2920.15593DOI Listing
May 2021

Interactions between Rainbow Trout Eyed Eggs and spp. Using a Bath Challenge Model: Preliminary Evaluation of Bacteriophages as Pathogen Control Agents.

Microorganisms 2021 Apr 30;9(5). Epub 2021 Apr 30.

Unit for Fish and Shellfish Diseases, National Institute of Aquatic Resources, Technical University of Denmark, 2800 Kongens Lyngby, Denmark.

The microbial community surrounding fish eyed eggs can harbor pathogenic bacteria. In this study we focused on rainbow trout () eyed eggs and the potential of bacteriophages against the pathogenic bacteria and . An infection bath method was first established, and the effects of singular phages on fish eggs was assessed (survival of eyed eggs, interaction of phages with eyed eggs). Subsequently, bacteria-challenged eyed eggs were exposed to phages to evaluate their effects in controlling the bacterial population. Culture-based methods were used to enumerate the number of bacteria and/or phages associated with eyed eggs and in the surrounding environment. The results of the study showed that, with our infection model, it was possible to re-isolate associated with eyed eggs after the infection procedure, without affecting the survival of the eggs in the short term. However, this was not possible for , as this bacterium grows at higher temperatures than the ones recommended for incubation of rainbow trout eyed eggs. Bacteriophages do not appear to negatively affect the survival of rainbow trout eyed eggs and they do not seem to strongly adhere to the surface of eyed eggs either. Finally, the results demonstrated a strong potential for short term (24 h) phage control of . However, further studies are needed to explore if phage control can be maintained for a longer period and to further elucidate the mechanisms of interactions between Flavobacteria and their phages in association with fish eggs.
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http://dx.doi.org/10.3390/microorganisms9050971DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8146780PMC
April 2021

Phage-Mediated Control of in Aquaculture: Experiments to Compare Delivery Methods.

Front Microbiol 2021 8;12:628309. Epub 2021 Mar 8.

Unit for Fish and Shellfish Diseases, National Institute of Aquatic Resources, Technical University of Denmark, Kgs. Lyngby, Denmark.

Phage-based approaches have gained increasing interest as sustainable alternative strategies to antibiotic treatment or as prophylactic measures against disease outbreaks in aquaculture. The potential of three methods (oral, bath, and injection) for delivering a two-component phage mixture to rainbow trout fry for controlling infections and reduce fish mortality was investigated using bacteriophages FpV4 and FPSV-D22. For the oral administration experiment, bacteriophages were applied on feed pellets by spraying (1.6 × 10 PFU g) or by irreversible immobilization (8.3 × 10 PFU g), using the corona discharge technology (Fixed Phage Ltd.). The fish showed normal growth for every group and no mortality was observed prior to infection as well as in control groups during the infection. Constant detection of phages in the intestine (∼10 PFU mg) and more sporadic occurrence in kidney, spleen, and brain was observed. When fish were exposed to , no significant effect on fish survival, nor a direct impact on the number of phages in the sampled organs, were detected. Similarly, no significant increase in fish survival was detected when phages were delivered by bath (1 and 2 bath: ∼10 PFU ml; 3 bath: ∼10 PFU ml). However, when phages FpV4 and FPSV-D22 (1.7 × 10 PFU fish) were administered by intraperitoneal injection 3 days after the bacterial challenge, the final percent survival observed in the group injected with bacteriophages FpV4 and FPSV-D22 (80.0%) was significantly higher than in the control group (56.7%). The work demonstrates the delivery of phages to fish organs by oral administration, but also suggests that higher phage dosages than the tested ones may be needed on feed pellets to offer fish an adequate protection against infections.
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http://dx.doi.org/10.3389/fmicb.2021.628309DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7983945PMC
March 2021

A Major QTL for Resistance to in Rainbow Trout.

Front Genet 2020 29;11:607558. Epub 2020 Dec 29.

Laboratory of Aquatic Pathobiology, Department of Veterinary and Animal Sciences, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark.

Genetic selection of disease resistant fish is a major strategy to improve health, welfare and sustainability in aquaculture. Mapping of single nucleotide polymorphisms (SNP) in the fish genome may be a fruitful tool to define relevant quantitative trait loci (QTL) and we here show its use for characterization of resistant rainbow trout (). Fingerlings were exposed to the pathogen serotype O1 in a solution of 1.5 × 10 cfu/ml and observed for 14 days. Disease signs appeared 3 days post exposure (dpe) whereafter mortality progressed exponentially until 6 dpe reaching a total mortality of 55% within 11 days. DNA was sampled from all fish - including survivors - and analyzed on a 57 k Affymetrix SNP platform whereby it was shown that disease resistance was associated with a major QTL on chromosome 21 (Omy 21). Gene expression analyses showed that diseased fish activated genes associated with innate and adaptive immune responses. The possible genes associated with resistance are discussed.
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http://dx.doi.org/10.3389/fgene.2020.607558DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7802751PMC
December 2020

Analysis of 44 Vibrio anguillarum genomes reveals high genetic diversity.

PeerJ 2020 3;8:e10451. Epub 2020 Dec 3.

National Institute of Aquatic Resources Technical University of Denmark, Kongens Lyngby, Denmark.

Vibriosis, a hemorrhagic septicemic disease caused by the bacterium , is an important bacterial infection in Danish sea-reared rainbow trout. Despite of vaccination, outbreaks still occur, likely because the vaccine is based on strains from abroad/other hosts than rainbow trout. Information about the genetic diversity of specifically in Danish rainbow trout, is required to investigate this claim. Consequently, the aim of the present investigation was to sequence and to characterize a collection of 44 strains obtained primarily from vibriosis outbreaks in Danish rainbow trout. The strains were sequenced, de novo assembled, and the genomes examined for the presence of plasmids, virulence, and acquired antibiotic resistance genes. To investigate the phylogeny, single nucleotide polymorphisms were identified, and the pan-genome was calculated. All strains carried encoding tetracycline resistance, and 36 strains also contained for increased fluoroquinolone/quinolone resistance. But interestingly, all strains were phenotypic sensitive to both oxytetracycline and oxolinic acid. Almost all serotype O1 strains contained a pJM1-like plasmid and nine serotype O2A strains carried the plasmid p15. The distribution of virulence genes was rather similar across the strains, although evident variance among serotypes was observed. Most significant, almost all serotype O2 and O3 strains, as well as the serotype O1 strain without a pJM1-like plasmid, carried genes encoding piscibactin biosynthesis. Hence supporting the hypothesis, that piscibactin plays a crucial role in virulence for pathogenic strains lacking the anguibactin system. The phylogenetic analysis and pan-genome calculations revealed great diversity within . Serotype O1 strains were in general very similar, whereas considerable variation was found among serotype O2A strains. The great diversity within the serotype O2A genomes is most likely the reason why vaccines provide good protection from some strains, but not from others. Hopefully, the new genomic data and knowledge provided in this study might help develop an optimized vaccine against in the future to reduce the use of antibiotics, minimize economic losses and improve the welfare of the fish.
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http://dx.doi.org/10.7717/peerj.10451DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7719292PMC
December 2020

Immune gene expression and genome-wide association analysis in rainbow trout with different resistance to Yersinia ruckeri infection.

Fish Shellfish Immunol 2020 Nov 11;106:441-450. Epub 2020 Aug 11.

Department of Veterinary and Animal Sciences, Faculty of Health and Medical Sciences, University of Copenhagen, Frederiksberg C., Denmark.

Selective breeding programmes involving marker assisted selection of innately pathogen resistant strains of rainbow trout rely on reliable controlled infection studies, extensive DNA typing of individual fish and recording of expression of relevant genes. We exposed juvenile rainbow trout (6 h bath to 2.6 × 10 CFU mL) to the fish pathogen Yersinia ruckeri serotype O1, biotype 2, eliciting Enteric Red Mouth Disease ERM, and followed the disease progression over 21 days. Cumulative mortality reached 42% at 12 days post challenge (dpc) after which no disease signs were recorded. All fish were sampled for DNA-typing (50 k SNP chip, Affymetrix®) throughout the course of infection when they showed clinical signs of disease (susceptible fish) or at day 21 when fish showed no clinical signs of disease (survivors - resistant fish). Genome-wide association analyses of 1027 trout applying single nucleotide polymorphisms (SNPs) as markers revealed an association between traits (susceptible/resistant) and certain regions of the trout genome. It was indicated that multiple genes are involved in rainbow trout resistance towards ERM whereby it is considered a polygenic trait. A corresponding trout group was kept as non-exposed controls and a comparative expression analysis of central innate and adaptive immune genes in gills, spleen and liver was performed for three fish groups: 1) moribund trout exhibiting clinical signs 7 dpc (CS), 2) exposed fish without clinical signs at the same sampling point (NCS) and 3) surviving fish at 21 dpc (survivors). Immune genes encoding inflammatory cytokines (IL-1β, IL-2A, IL-6A, IL-8, IL-10A, IL-12, IL-17A/F2A, IL-17C1, IL-17C2, IL-22, IFNγ, TNFα), acute phase reactants (SAA, C3, cathelicidins, lysozyme) were expressed differently in CS and NCS fish. Correlation (negative or positive) between expression of genes and bacterial load suggested involvement of immune genes in protection. Down-regulation of adaptive immune genes including IgDm, IgDs, IgT and TCR-β was seen primarily in CS and NCS fish whereas survivors showed up-regulation of effector molecule genes such as cathelicidins, complement and lysozyme suggesting their role in clearing the infection. In conclusion, SNP analyses indicated that ERM resistance in rainbow trout is a multi-locus trait. The gene expression in surviving fish suggested that several immune genes are associated with the trait conferring resistance.
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http://dx.doi.org/10.1016/j.fsi.2020.07.023DOI Listing
November 2020

Sequence Analysis of Plasmids in Vibrio anguillarum from Different Fish and Locations.

J Aquat Anim Health 2020 03 27;32(1):21-27. Epub 2020 Jan 27.

National Veterinary Institute, Technical University of Denmark, Anker Engelunds Vej 1, DK-2800, Kongens Lyngby, Denmark.

The genetic diversity of Vibrio anguillarum pJM1-like plasmids was investigated. Plasmids were isolated from 18 V. anguillarum serovar O1 strains collected from different geographic locations and fish species. The plasmids were sequenced and compared with the complete sequence of the published virulence plasmid pJM1. All 18 strains contained pJM1-like plasmids with approximately 65 kbp and all plasmids encoded the virulence genes responsible for the anguibactin iron sequestering system. The plasmids were highly conserved but minor differences were observed in some genes. A single nucleotide polymorphisms (SNPs) analysis showed 0-11 nucleotide variations between each of the 18 plasmids and the pJM1 plasmid. Compared with the sequence of pJM1, nonsynonymous SNPs were identified in fatC, angR, angL, pJM1_p19, and angE. In particular, a mutation found in 15 out of 18 sequenced plasmids in angR has previously been linked to hyperproduction of anguibactin and was found in all the European isolates. However, overall the pJM1-like plasmids isolated from V. anguillarum serovar O1 exhibited a high degree of conservation regardless of their geographical origin or fish species.
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http://dx.doi.org/10.1002/aah.10093DOI Listing
March 2020

Protective effect of in-feed specific IgM towards Yersinia ruckeri in rainbow trout.

Fish Shellfish Immunol 2019 Oct 10;93:934-939. Epub 2019 Aug 10.

National Veterinary Institute, Technical University of Denmark, Frederiksberg C, Denmark. Electronic address:

Tightened regulations and an environmentally friendly approaches in fish production have greatly reduced the use of antibiotics but green solutions are continuously being explored. The use of functional feed may have a potential in the aquaculture sector in securing biomass and minimizing the loss from disease. In the present study, we tested the concept that blood from the fish slaughterhouse can be used for mass purification of specific antibodies which subsequently can be used for feeding fish and thereby confer protection against diseases. IgM was purified from serum from Yersinia ruckeri vaccinated rainbow trout and an IgM sandwich ELISA was developed for quantification of rainbow trout IgM. The purified IgM was encapsulated in alginate microparticles and top-coated in fish feed. IgM re-extracted from the alginate microparticles was shown to retain high reactivity towards Y. ruckeri antigens indicating that its bioactivity remained intact after encapsulation. IgM release from the alginate microparticles was only observed at high pH (pH 8.2) and minimal at low pH, indicating protection of IgM at low pH in the fish stomach during passage. In a feeding - challenge experiment (feeding 1 week before Y. ruckeri challenge and for two weeks following challenge), a statistically non-significant 10% lower mortality was observed in the high dose (400 μg IgM/fish/day fed over 3 weeks) group.
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http://dx.doi.org/10.1016/j.fsi.2019.08.024DOI Listing
October 2019

Phenotypic and Genetic Predictors of Pathogenicity and Virulence in .

Front Microbiol 2019 24;10:1711. Epub 2019 Jul 24.

Laboratory of Aquatic Pathobiology, Environmental and Marine Biology, Åbo Akademi University, Turku, Finland.

causes bacterial cold-water disease (BCWD) in farmed rainbow trout (), with the multilocus sequence typing (MLST) clonal complex (CC) CC-ST10 accounting for the majority of outbreaks globally. The development of alternative strategies to antibiotic treatment of BCWD using bacteriophage-based control of , or virulence factors as targets for therapy, requires knowledge of the phage-sensitivity of outbreak strains and of universal traits contributing to their pathogenicity. To examine the association between virulence and both genetic (MLST sequence type (ST) and PCR-serotype) and phenotypic characteristics (adherence, antibiotic resistance, colony spreading motility, hemolytic and proteolytic activity), the median lethal dose (LD) of 26 geographically disparate isolates was determined in rainbow trout. Furthermore, the sensitivity of the isolates against five bacteriophages was determined by the efficiency of plating (EOP). The tested isolates were mainly represented by CC-ST10 genotypes (22 out of 26) and showed up to 3-log differences in LD (8.9 × 10 to 3.1 × 10 CFU). No association between MLST ST and virulence was found because of a high variation in LD within STs. All identified serotypes (0, 1, and 2) were pathogenic, but ten most virulent isolates belonged to serotype 1 or 2. Isolates of high (LD < 10 CFU), moderate (LD = 10-10 CFU), and weak (LD > 10 CFU) virulence were similar in phenotypic characteristics . However, the only non-virulent CC-ST10 isolate was deficient in spreading motility and proteolytic activity, indicating that the characteristics are required for pathogenicity in . Univariate correlation studies found only non-significant associations between LD and the measured phenotypic characteristics, and the multivariable analysis did neither reveal any significant predictors of virulence. The majority of isolates (16 out of 26) were sensitive to at least four bacteriophages, with up to a 6-log variation in the EOP. Most CC-ST10 isolates (16 out of 22) were sensitive to the examined phages, including 5 out of the 7 most virulent isolates represented by prevalent and antibiotic-resistant STs. Our findings suggest that control of BCWD using lytic phages or interventions targeting shared characteristics of pathogenic strains should be further explored.
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http://dx.doi.org/10.3389/fmicb.2019.01711DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6668605PMC
July 2019

Inhibition Activity of Compounds and Bacteriophages against Flavobacterium psychrophilum Biofilms In Vitro.

J Aquat Anim Health 2019 09 19;31(3):225-238. Epub 2019 Jun 19.

Laboratory of Aquatic Pathobiology, Environmental and Marine Biology, Åbo Akademi University, Tykistokatu 6, FI-20520, Turku, Finland.

Flavobacterium psychrophilum produces biofilms under laboratory conditions, and it has been inconclusively suggested that F. psychrophilum biofilms can be a potential reservoir for transmission of the pathogen to a fish population under fish farming conditions. Therefore, there is a need for anti-biofilm compounds. The main aim of this study was to determine the anti-biofilm properties of certain compounds and bacteriophages on F. psychrophilum biofilms under static conditions using a standard 96-well microtiter plate biofilm assay in vitro. Eight compounds (A-type proanthocyanidins, D-leucine, EDTA, emodin, fucoidan, L-alliin, parthenolide, and 2-aminoimidazole) at three sub-minimum inhibitory concentrations (sub-MICs), four bacteriophages (Fpv-3, Fpv-9, Fpv-10, and Fpv-21), and a phage combination (Fpv-9 + Fpv-10) were tested for inhibition of biofilm formation and reduction of the biomass of mature biofilms formed by two smooth isolates (P7-9/10 and P1-10B/10) and two rough isolates (P7-9/2R/10 and P1-10B/2R/10) of F. psychrophilum. The crystal violet staining method was used to stain the biofilms. Most of the compounds at sub-MICs inhibited the biofilm formation of mainly smooth isolates, attaining up to 80% inhibition. Additionally, the same reduction trend was also observed for 2-aminoimidazole, emodin, parthenolide, and D-leucine on the biomass of mature biofilms in a concentration-dependent manner. The anti-biofilm properties of the compounds are believed to lie in their ability to disturb the cellular interactions during biofilm formation and probably to cause cell dispersal in already formed biofilms. Lytic bacteriophages efficiently inhibited biofilm formation of F. psychrophilum, while they partially reduced the biomass of mature biofilms. However, the phage combination (Fpv-9 + Fpv-10) showed a successful reduction in the biomass of F. psychrophilum mature biofilms. We conclude that inhibiting compounds together with bacteriophages may supplement the use of disinfectants against bacterial biofilms (e.g., F. psychrophilum biofilms), leading to a reduced occurrence of bacterial coldwater disease outbreaks at fish farms.
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http://dx.doi.org/10.1002/aah.10069DOI Listing
September 2019

Biogeography of the fish pathogen Aeromonas salmonicida inferred by vapA genotyping.

FEMS Microbiol Lett 2019 04;366(7)

Fish Health Research Group, Norwegian Veterinary Institute, Oslo, Norway.

A recently described typing system based on sequence variation in the virulence array protein (vapA) gene, encoding the A-layer surface protein array, allows unambiguous subtyping of Aeromonas salmonicida. In the present study, we compile A-layer typing results from a total of 675 A. salmonicida isolates, recovered over a 59-year period from 50 different fish species in 26 countries. Nine novel A-layer types (15-23) are identified, several of which display a strong predilection towards certain fish hosts, including e.g. Cyprinidae and Pleuronectidae species. Moreover, we find indications that anthropogenic transport of live fish may have aided the near global dissemination of two cyprinid-associated A-layer types. Comparison of whole genome phylogeny and A-layer typing for a subset of strains further resulted in compatible tree topologies, indicating the utility of vapA as a phylogenetic as well as an epizootiological marker in A. salmonicida. A Microreact project (microreact.org/project/r1pcOAx9m) has been created, allowing public access to the vapA analyses and relevant metadata. In sum, the results generated provide valuable insights into the global population structure of A. salmonicida, particularly in relation to its piscine host spectrum and the geographic distribution of these hosts.
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http://dx.doi.org/10.1093/femsle/fnz074DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6502549PMC
April 2019

A pentavalent vaccine for rainbow trout in Danish aquaculture.

Fish Shellfish Immunol 2019 May 6;88:344-351. Epub 2019 Mar 6.

Department of Veterinary and Animal Science, Faculty of Health and Medical Sciences, University of Copenhagen, Denmark.

Mariculture in Denmark is based on production of rainbow trout grown two years in fresh water followed by one growth season in sea cages. Although the majority of rainbow trout are vaccinated against the most serious bacterial pathogens - Aeromonas salmonicida subsp. salmonicida, Vibrio anguillarum and Yersinia ruckeri, by the use of commercially available vaccines, disease outbreaks requiring treatment with antibiotics still occur. The present study tested the potential of a new experimental multicomponent vaccine that is based on local bacterial strains, isolated from rainbow trout in Danish waters, and thus custom-designed for Danish rainbow trout mariculture. The vaccination with the multicomponent vaccine resulted in protection against three relevant bacterial diseases (yersiniosis, furunculosis, vibriosis) under experimental conditions. We showed that i.p. injection of the vaccine induced specific antibody responses in trout against the different bacterial antigens and regulated expression of genes encoding SAA, C3, IL-1β, IL-6, IL-8, IgD and MHCII.
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http://dx.doi.org/10.1016/j.fsi.2019.03.001DOI Listing
May 2019

Molecular Epidemiology of subsp. Outbreaks in Marine Rainbow Trout Farms Reveals Extensive Horizontal Gene Transfer and High Genetic Diversity.

Front Microbiol 2018 19;9:2155. Epub 2018 Sep 19.

Departamento de Microbioloxía e Parasitoloxía, Instituto de Acuicultura, Universidade de Santiago de Compostela, Santiago de Compostela, Spain.

The marine bacterium subsp. is a pathogen for a variety of marine animals, as well as for humans, and is nowadays considered an emerging pathogen for fish of importance in marine aquaculture. Recent studies have suggested that outbreaks in fish farms are caused by multiclonal populations of this subspecies that exist in the environment. Here, we report the study of a collection of 31 strains isolated during the course of disease outbreaks in marine rainbow trout farms in Denmark in 1994, 1995, and 2006, respectively. A phylogenetic analysis based on the gene sequence, and the screening of virulence-related genes uncovered a high genetic heterogeneity, even among strains isolated from the same fish farm at the same time. Moreover, comparative analysis of the whole genome sequences of four selected strains revealed a large number of differentially occurring genes, which included virulence genes, pPHDD1 plasmid, polysaccharide synthesis gene clusters, CRISPR-Cas systems and putative new mobile genetic elements. This study provides sound evidence that subsp. outbreaks in Danish rainbow trout farms were caused by multiclonal populations and that horizontal gene transfer constitutes a strong driving force in the generation of intraspecific diversity in this pathogen.
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http://dx.doi.org/10.3389/fmicb.2018.02155DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6156455PMC
September 2018

Experimental anal infection of rainbow trout with Flavobacterium psychrophilum: A novel challenge model.

J Fish Dis 2018 Dec 11;41(12):1917-1919. Epub 2018 Sep 11.

Department of Veterinary and Animal Science, Faculty of Health and Medical Sciences, University of Copenhagen, Frederiksberg C, Denmark.

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http://dx.doi.org/10.1111/jfd.12888DOI Listing
December 2018

Genomic Diversity and Evolution of the Fish Pathogen .

Front Microbiol 2018 7;9:138. Epub 2018 Feb 7.

Unité Mathématiques et Informatique Appliquées du Génome à l'Environnement (MaIAGE), Institut National de la Recherche Agronomique, Université Paris-Saclay, Jouy-en-Josas, France.

, 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 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 () 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 , 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.
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http://dx.doi.org/10.3389/fmicb.2018.00138DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5808330PMC
February 2018

Epidemiology of Danish subsp. in Fish Farms Using Whole Genome Sequencing.

Front Microbiol 2017 5;8:2411. Epub 2017 Dec 5.

Section for Bacteriology and Pathology, National Veterinary Institute, Technical University of Denmark, Lyngby, Denmark.

Furunculosis, a serious infection caused by the bacterium subsp. is common in sea-reared rainbow trout production in Denmark. Developing an effective control strategy requires knowledge of the epidemiology, as well as the genomic and virulent variability of the Danish subsp. isolates. To obtain this, the genomes of 101 subsp. , including 99 Danish isolates, one Scottish strain and the type strain NCIMB 1102, were sequenced using the Illumina HiSeq platform. Isolates were assembled, examined for presence of plasmids, virulence and iron acquisition proteins, genomic islands, and antibiotic resistance genes. Single Nucleotide Polymorphisms were aligned and subjected to Bayesian temporal phylogenetic and maximum likelihood tree reconstruction using the published genome of subsp. A449 as reference. Bayesian temporal phylogenetic reconstruction suggests that four major introductions of subsp. into Denmark have occurred. The introductions correlate with the freshwater and subsequent seawater expansion of rainbow trout production. Initial transmission of the bacterium could have been from seawater to freshwater or vice versa, and most minor clades include a mixture of strains from different fresh- and seawater farms. Genomic variation of subsp. mostly appeared to be associated with their plasmids and plasmid encoded virulence factors. Nine subsp. isolates harbored worldwide known antibiotic resistance genes against several antibiotics and there is an indication that 33% of the isolates contained the genomic island AsaGEI1b. These findings not only support the usefulness of whole genome sequencing for genetic studies of homogeneous bacteria in general, but provide novel information about the Danish subsp. population, with implications for vaccine development in efforts to better protect Danish rainbow trout in the future.
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http://dx.doi.org/10.3389/fmicb.2017.02411DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5723325PMC
December 2017

Effect of oral booster vaccination of rainbow trout against Yersinia ruckeri depends on type of primary immunization.

Fish Shellfish Immunol 2019 Feb 31;85:61-65. Epub 2017 Oct 31.

Department of Veterinary and Animal Science, Faculty of Health and Medical Sciences, University of Copenhagen, Frederiksberg C, Denmark.

Vaccination of rainbow trout against Enteric Redmouth Disease (ERM) caused by Yersinia ruckeri can be successfully performed by administering vaccine (a bacterin consisting of formalin killed bacteria) by immersion, bath or injection. Booster immunization is known to increase the protection of fish already primed by one of these vaccination methods. Oral vaccination of trout (administering vaccine in feed) is an even more convenient way of presenting antigen to the fish but the effect of an oral booster has not previously been described in detail. The present work describes to what extent protection may be enhanced by oral boostering following priming with different administration methods. The study confirms that vaccination by 30 s dip into a bacterin (diluted 1:10) may confer a significant protection compared to non-vaccinated fish. The immunity may be optimized by booster immunization either provided as dip (most effective), bath (less effective) or orally (least effective). Oral immunization may be used as booster after dip but applied as a single oral application it induced merely a slight and statistically non-significant response. It is noteworthy that primary oral immunization followed by an oral booster vaccination showed a trend for an even weaker response. It should be investigated if continued exposure to a low antigen concentration - as performed by two oral immunizations - may induce tolerance to the pathogen and thereby leave the fish more vulnerable.
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http://dx.doi.org/10.1016/j.fsi.2017.10.049DOI Listing
February 2019

Genomic Characterization of Serotypes and Development of a Multiplex PCR-Based Serotyping Scheme.

Front Microbiol 2017 12;8:1752. Epub 2017 Sep 12.

Virologie et Immunologie Moléculaires, Institut National de la Recherche Agronomique, Université Paris-SaclayJouy-en-Josas, France.

is a devastating bacterial pathogen of salmonids reared in freshwater worldwide. So far, serological diversity between isolates has been described but the underlying molecular factors remain unknown. By combining complete genome sequence analysis and the serotyping method proposed by Lorenzen and Olesen (1997) for a set of 34 strains, we identified key molecular determinants of the serotypes. This knowledge allowed us to develop a robust multiplex PCR-based serotyping scheme, which was applied to 244 bacterial isolates. The results revealed a striking association between PCR-serotype and fish host species and illustrate the use of this approach as a simple and cost-effective method for the determination of serogroups. PCR-based serotyping could be a useful tool in a range of applications such as disease surveillance, selection of salmonids for bacterial coldwater disease resistance and future vaccine formulation.
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http://dx.doi.org/10.3389/fmicb.2017.01752DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5601056PMC
September 2017

Subunit vaccine candidates against Aeromonas salmonicida in rainbow trout Oncorhynchus mykiss.

PLoS One 2017 9;12(2):e0171944. Epub 2017 Feb 9.

Department of Veterinary Disease Biology, Faculty of Health and Medical Sciences, University of Copenhagen, Denmark.

Aeromonas salmonicida subsp. salmonicida is the etiological agent of furunculosis and a major fish health problem in salmonid aquaculture worldwide. Injection vaccination with commercial mineral oil-adjuvanted bacterin vaccines has been partly successful in preventing the disease but in Danish rainbow trout (Oncorhynchus mykiss, Walbaum) aquaculture furunculosis outbreaks still occur. In this study we tested the efficacy of experimental subunit vaccines against A. salmonicida infection in rainbow trout. We utilized in silico screening of the proteome of A. salmonicida subsp. salmonicida strain A449 and identified potential protective protein antigens that were tested by in vivo challenge trial. A total of 14 proteins were recombinantly expressed in Escherichia coli and prepared in 3 different subunit vaccine combinations to immunize 3 groups of rainbow trout by intraperitoneal (i.p.) injection. The fish were exposed to virulent A. salmonicida 7 weeks after immunization. To assess the efficacy of the subunit vaccines we evaluated the immune response in fish after immunization and challenge infection by measuring the antibody levels and monitoring the survival of fish in different groups. The survival of fish at 3 weeks after challenge infection showed that all 3 groups of fish immunized with 3 different protein combinations exhibited significantly lower mortalities (17-30%) compared to the control groups (48% and 56%). The ELISA results revealed significantly elevated antibody levels in fish against several protein antigens, which in some cases were positively correlated to the survival.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0171944PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5300222PMC
August 2017

Antigen Uptake during Different Life Stages of Zebrafish (Danio rerio) Using a GFP-Tagged Yersinia ruckeri.

PLoS One 2016 12;11(7):e0158968. Epub 2016 Jul 12.

Laboratory of Aquatic Pathobiology, Department of Veterinary Disease Biology, University of Copenhagen, Frederiksberg, Denmark.

Immersion-vaccines (bacterins) are routinely used for aquacultured rainbow trout to protect against Yersinia ruckeri (Yr). During immersion vaccination, rainbow trout take up and process the antigens, which induce protection. The zebrafish was used as a model organism to study uptake mechanisms and subsequent antigen transport in fish. A genetically modified Yr was developed to constitutively express green fluorescent protein (GFP) and was used for bacterin production. Larval, juvenile and adult transparent zebrafish (tra:nac mutant) received a bath in the bacterin for up to 30 minutes. Samples were taken after 1 min, 15 min, 30 min, 2 h, 12 h and 24 h. At each sampling point fish were used for live imaging of the uptake using a fluorescence stereomicroscope and for immunohistochemistry (IHC). In adult fish, the bacterin could be traced within 30 min in scale pockets, skin, oesophagus, intestine and fins. Within two hours post bath (pb) Yr-antigens were visible in the spleen and at 24 h in liver and kidney. Bacteria were associated with the gills, but uptake at this location was limited. Antigens were rarely detected in the blood and never in the nares. In juvenile fish uptake of the bacterin was seen in the intestine 30 min pb and in the nares 2 hpb but never in scale pockets. Antigens were detected in the spleen 12 hpb. Zebrafish larvae exhibited major Yr uptake only in the mid-intestine enterocytes 24 hpb. The different life stages of zebrafish varied with regard to uptake locations, however the gut was consistently a major uptake site. Zebrafish and rainbow trout tend to have similar uptake mechanisms following immersion or bath vaccination, which points towards zebrafish as a suitable model organism for this aquacultured species.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0158968PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4942034PMC
July 2017

Comparative Genome Analysis Provides Insights into the Pathogenicity of Flavobacterium psychrophilum.

PLoS One 2016 12;11(4):e0152515. Epub 2016 Apr 12.

Marine Biological Section, University of Copenhagen, Helsingør, Denmark.

Flavobacterium psychrophilum is a fish pathogen in salmonid aquaculture worldwide that causes cold water disease (CWD) and rainbow trout fry syndrome (RTFS). Comparative genome analyses of 11 F. psychrophilum isolates representing temporally and geographically distant populations were used to describe the F. psychrophilum pan-genome and to examine virulence factors, prophages, CRISPR arrays, and genomic islands present in the genomes. Analysis of the genomic DNA sequences were complemented with selected phenotypic characteristics of the strains. The pan genome analysis showed that F. psychrophilum could hold at least 3373 genes, while the core genome contained 1743 genes. On average, 67 new genes were detected for every new genome added to the analysis, indicating that F. psychrophilum possesses an open pan genome. The putative virulence factors were equally distributed among isolates, independent of geographic location, year of isolation and source of isolates. Only one prophage-related sequence was found which corresponded to the previously described prophage 6H, and appeared in 5 out of 11 isolates. CRISPR array analysis revealed two different loci with dissimilar spacer content, which only matched one sequence in the database, the temperate bacteriophage 6H. Genomic Islands (GIs) were identified in F. psychrophilum isolates 950106-1/1 and CSF 259-93, associated with toxins and antibiotic resistance. Finally, phenotypic characterization revealed a high degree of similarity among the strains with respect to biofilm formation and secretion of extracellular enzymes. Global scale dispersion of virulence factors in the genomes and the abilities for biofilm formation, hemolytic activity and secretion of extracellular enzymes among the strains suggested that F. psychrophilum isolates have a similar mode of action on adhesion, colonization and destruction of fish tissues across large spatial and temporal scales of occurrence. Overall, the genomic characterization and phenotypic properties may provide new insights to the mechanisms of pathogenicity in F. psychrophilum.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0152515PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4829187PMC
August 2016

Effect of Bacteriophages on the Growth of Flavobacterium psychrophilum and Development of Phage-Resistant Strains.

Microb Ecol 2016 May 22;71(4):845-59. Epub 2016 Feb 22.

Marine Biological Section, University of Copenhagen, Strandpromenaden 5, 3000, Helsingør, Denmark.

The controlling effect of single and multiple phages on the density of Flavobacterium psychrophilum at different initial multiplicity of infection (MOI) was assessed in batch cultures to explore the potential for phage-based treatment of this important fish pathogen. A high initial phage concentration (MOI = 0.3-4) was crucial for efficient viral lysis, resulting in a 10(4)-10(5)-fold reduction of phage-sensitive cells (both single phages and phage cocktails), which was maintained throughout the incubation (>10 days). Following cell lysis, regrowth of phage-resistant strains was examined and resistant strains were isolated for further characterization. The application of a mathematical model allowed simulation of phage-host interactions and resistance development, confirming indications from strain isolations that phage-sensitive strains dominated the regrowing population (>99.8%) at low MOI and phage-resistant strains (>87.8%) dominated at high MOI. A cross-infectivity test covering 68 isolated strains and 22 phages resulted in 23 different host susceptibility patterns, with 20 of the isolates being resistant to all the applied phages. Eleven isolated strains with different susceptibility patterns had lower growth rates (0.093 to 0.31 h(-1)) than the host strain (0.33 h(-1)), while 10 of 14 examined strains had lost the ability to take up specific substrates as shown by BIOLOG profiles. Despite increased selection for phage resistance at high MOI, the results emphasize that high initial MOI is essential for fast and effective control of F. psychrophilum infection and suggest that the small populations of resistant clones had reduced competitive abilities relative to the sensitive ancestral strain.
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http://dx.doi.org/10.1007/s00248-016-0737-5DOI Listing
May 2016

Effects of adjuvant Montanide™ ISA 763 A VG in rainbow trout injection vaccinated against Yersinia ruckeri.

Fish Shellfish Immunol 2015 Dec 19;47(2):797-806. Epub 2015 Oct 19.

Department of Veterinary Disease Biology, Faculty of Health and Medical Sciences, University of Copenhagen, Frederiksberg C, Denmark.

Enteric redmouth disease (ERM) caused by the fish pathogen Yersinia ruckeri is a major threat to freshwater production of rainbow trout (Oncorhynchus mykiss) throughout all life stages. Injection vaccination of rainbow trout against Y. ruckeri infection has been shown to confer better protection compared to the traditionally applied immersion vaccination. It may be hypothesized, based on experience from other vaccines, that adjuvants may increase the protective level of ERM injection vaccines even more. Controlled comparative vaccination studies have been performed to investigate effects of the oil adjuvant Montanide™ ISA 763 A VG (Seppic) when added to an experimental Y. ruckeri bacterin (containing both biotype 1 and 2 of serotype O1). A total of 1000 fish with mean weight 19 g was divided into five different groups (in duplicated tanks 2 × 100 fish per group) 1) non-vaccinated control fish (NonVac), 2) fish injected with a commercial vaccine (AquaVac(®) Relera™) (ComVac), 3) fish injected with an experimental vaccine (ExpVac), 4) fish injected with an experimental vaccine + adjuvant (ExpVacAdj) and 5) fish injected with adjuvant alone (Adj). Injection of the experimental vaccine (both adjuvanted and non-adjuvanted) induced a significantly higher antibody (IgM) level, increased occurrence of IgM(+) cells in spleen tissue and significant up-regulation of several immune genes. Additional experiments using a higher challenge dosage suggested an immune enhancing effect of the adjuvant as the challenge produced 100% mortality in the NonVac group, 60% mortality in both of ComVac and Adj groups and only 13 and 2.5% mortalities in the ExpVac and the ExpVacAdj groups, respectively.
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http://dx.doi.org/10.1016/j.fsi.2015.10.023DOI Listing
December 2015

Variable Number of Tandem Repeats (VNTR) analysis of Flavobacterium psychrophilum from salmonids in Chile and Norway.

BMC Vet Res 2015 Jul 14;11:150. Epub 2015 Jul 14.

Fish Diseases Research Group, Department of Biology, University of Bergen, P.O. 7803, N-5020, Bergen, Norway.

Background: Flavobacterium psychrophilum causes serious fish diseases such RTFS and BCWD, affecting the aquaculture industry worldwide. Commercial vaccines are not available and control of the disease depends on the use of antibiotics. Reliable methods for detection and identification of different isolates of this bacterium could play an important role in the development of good management strategies. The aim of this study was to identify genetic markers for discrimination between isolates. A selection of eight VNTRs from 53 F. psychrophilum isolates from Norway, Chile, Denmark and Scotland were analyzed. The results were compared with previous work on the same pathogen using MLST for genetic differentiation.

Results: The VNTR analysis gave a separation between the F. psychrophilum isolates supporting the results of previous MLST work. A higher diversity was found among the Chilean isolates compared to those from Norway, which suggests a more homogenous reservoir in Norway. Transgenerational transmission of F. psychrophilum from other countries, exporting salmon embryos to Chile, may explain the differences in diversity. The same transmission mechanisms could also explain the wide geographical distribution of identical isolates in Norway. But, this could also be a result of movement of smolts and embryos. The selected VNTRs are stable genetic markers and no variation was observed after several passages on agar plates at different temperatures.

Conclusions: These VNTRs are important additions for genotyping of F. psychrophilum isolates. Future studies on VNTRs of F. psychrophilum should include isolates from more host species from a wider geographical area. To get a more robust genotyping the VNTRs should be used in concert with MLST. Future studies of isolates with high and low virulence should focus on identifying virulence markers using VTNRs and MLST.
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http://dx.doi.org/10.1186/s12917-015-0469-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4501049PMC
July 2015

Comparative evaluation of infection methods and environmental factors on challenge success: Aeromonas salmonicida infection in vaccinated rainbow trout.

Fish Shellfish Immunol 2015 Jun 14;44(2):485-95. Epub 2015 Mar 14.

Laboratory of Aquatic Pathobiology, Department of Veterinary Disease Biology, Faculty of Health and Medical Sciences, University of Copenhagen, Denmark. Electronic address:

When testing vaccine-induced protection an effective and reliable challenge method is a basic requirement and we here present a comparative study on different challenge methods used for infection of rainbow trout Oncorhynchus mykiss with Aeromonas salmonicida, a bacterial pathogen eliciting furunculosis. Fish were vaccinated with three different adjuvanted trivalent vaccines containing formalin killed A. salmonicida, Vibrio anguillarum O1 and O2a. These were 1) the commercial vaccine Alpha Ject 3000, 2) an experimental vaccine with water in paraffin oil adjuvant, 3) an experimental vaccine with water in paraffin oil in water adjuvant. Fish were then exposed to A. salmonicida challenge using i.p. injection, cohabitation in freshwater, cohabitation in saltwater (15 ppt) or combined fresh/saltwater cohabitation. Cohabitation reflects a more natural infection mode and was shown to give better differentiation of vaccine types compared to i.p. injection of live bacteria. The latter infection mode is less successful probably due to the intra-abdominal inflammatory reactions (characterized in this study according to the Speilberg scale) induced by i.p. vaccination whereby injected live bacteria more effectively become inactivated at the site of injection. Compared to cohabitation in freshwater, cohabitation in saltwater was less efficient probably due to reduced survivability of A. salmonicida in saltwater, which was also experimentally verified in vitro.
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http://dx.doi.org/10.1016/j.fsi.2015.03.003DOI Listing
June 2015

Bacteriophage resistance mechanisms in the fish pathogen Flavobacterium psychrophilum: linking genomic mutations to changes in bacterial virulence factors.

Appl Environ Microbiol 2015 Feb 5;81(3):1157-67. Epub 2014 Dec 5.

Marine Biological Section, University of Copenhagen, Helsingør, Denmark

Flavobacterium psychrophilum is an important fish pathogen in salmonid aquaculture worldwide. Due to increased antibiotic resistance, pathogen control using bacteriophages has been explored as a possible alternative treatment. However, the effective use of bacteriophages in pathogen control requires overcoming the selection for phage resistance in the bacterial populations. Here, we analyzed resistance mechanisms in F. psychrophilum after phage exposure using whole-genome sequencing of the ancestral phage-sensitive strain 950106-1/1 and six phage-resistant isolates. The phage-resistant strains had all obtained unique insertions and/or deletions and point mutations distributed among intergenic and genic regions. Mutations in genes related to cell surface properties, gliding motility, and biosynthesis of lipopolysaccharides and cell wall were found. The observed links between phage resistance and the genetic modifications were supported by direct measurements of bacteriophage adsorption rates, biofilm formation, and secretion of extracellular enzymes, which were all impaired in the resistant strains, probably due to superficial structural changes. The clustered regularly interspaced short palindromic repeat (CRISPR) region was unaffected in the resistant isolates and thus did not play a role as a resistance mechanism for F. psychrophilum under the current conditions. All together, the results suggest that resistance in F. psychrophilum was driven by spontaneous mutations, which were associated with a number of derived effects on the physiological properties of the pathogen, including reduced virulence under in vitro conditions. Consequently, phage-driven physiological changes associated with resistance may have implications for the impact of the pathogen in aquaculture, and these effects of phage resistance on host properties are therefore important for the ongoing exploration of phage-based control of F. psychrophilum.
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http://dx.doi.org/10.1128/AEM.03699-14DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4292493PMC
February 2015

Detection and quantification of Flavobacterium psychrophilum-specific bacteriophages in vivo in rainbow trout upon oral administration: implications for disease control in aquaculture.

Appl Environ Microbiol 2014 Dec 3;80(24):7683-93. Epub 2014 Oct 3.

National Veterinary Institute, Technical University of Denmark, Frederiksberg, Denmark

The use of bacteriophages in the treatment and prevention of infections by the fish pathogen Flavobacterium psychrophilum has attracted increased attention in recent years. It has been shown recently that phage delivery via the parenteral route resulted in immediate distribution of phages to the circulatory system and the different organs. However, little is known about phage dispersal and survival in vivo in rainbow trout after delivery via the oral route. Here we examined the dispersal and survival of F. psychrophilum phage FpV-9 in vivo in juvenile rainbow trout after administration by three different methods-bath, oral intubation into the stomach, and phage-coated feed-with special emphasis on the oral route of delivery. Phages could be detected in all the organs investigated (intestine, spleen, brain, and kidney) 0.5 h postadministration, reaching concentrations as high as ∼10(5) PFU mg intestine(-1) and ∼10(3) PFU mg spleen(-1) within the first 24 h following the bath and ∼10(7) PFU mg intestine(-1) and ∼10(4) PFU mg spleen(-1) within the first 24 h following oral intubation. The phages were most persistent in the organs for the first 24 h and then decreased exponentially; no phages were detected after 83 h in the organs investigated. Phage administration via feed resulted in the detection of phages in the intestine, spleen, and kidney 1 h after feeding. Average concentrations of ∼10(4) PFU mg intestine(-1) and ∼10(1) PFU mg spleen(-1) were found throughout the experimental period (200 h) following continuous delivery of phages with feed. These experiments clearly demonstrate the ability of the phages to survive passage through the fish stomach and to penetrate the intestinal barrier and enter the circulatory system after oral delivery, although the quantity of phages found in the spleen was 100- to 1,000-fold lower than that in the intestine. It was also shown that phages could tolerate long periods of desiccation on the feed pellets, with 60% survival after storage at -80°C, and 10% survival after storage at 5°C, for ∼8 months. Continuous delivery of phages via coated feed pellets constitutes a promising method of treatment and especially prevention of rainbow trout fry syndrome.
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http://dx.doi.org/10.1128/AEM.02386-14DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4249249PMC
December 2014

Diet type dictates the gut microbiota and the immune response against Yersinia ruckeri in rainbow trout (Oncorhynchus mykiss).

Fish Shellfish Immunol 2014 Oct 21;40(2):624-33. Epub 2014 Aug 21.

National Veterinary Institute, Technical University of Denmark, Bülowsvej 27, DK-1870 Frederiksberg C, Denmark. Electronic address:

This study investigated the influence of the rainbow trout (Oncorhynchus mykiss) commensal intestinal microbiota in connection to an experimental Yersina ruckeri infection, the causative agent of enteric redmouth disease. One marine and one plant diet was administered to two different groups of rainbow trout. The plant-based diet gave rise to an intestinal microbiota dominated by the genera Streptococcus, Leuconostoc and Weissella from phylum Firmicutes whereas phylum Proteobacteria/Bacteroidetes/Actinobacteria dominated the community in the marine fed fish. In connection to the Y. ruckeri bath challenge there was no effect of the diet type on the cumulative survival, but the number of Y. ruckeri positive fish as measured by plate count and the number of fish with a 'high' number of reads belonging to genus Yersinia as measured by 16S rRNA next-generation sequencing was higher for marine diet fed fish. Furthermore, the two experimental groups of fish showed a differential immune response, where Y. ruckeri challenged marine fed fish had a higher transcription of IL-1β and MBL-2 relative to challenged plant diet fed fish. The data suggest that the plant diet gave rise to a prebiotic effect favouring the presence of bacterial taxons proving protective in connection to bath challenge by Y. ruckeri.
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http://dx.doi.org/10.1016/j.fsi.2014.08.021DOI Listing
October 2014

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

Appl Environ Microbiol 2014 May 21;80(9):2728-36. Epub 2014 Feb 21.

Norwegian Veterinary Institute, Bergen, Norway.

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.
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http://dx.doi.org/10.1128/AEM.04233-13DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3993282PMC
May 2014

Insight from molecular, pathological, and immunohistochemical studies on cellular and humoral mechanisms responsible for vaccine-induced protection of rainbow trout against Yersinia ruckeri.

Clin Vaccine Immunol 2013 Oct 21;20(10):1623-41. Epub 2013 Aug 21.

Department of Veterinary Disease Biology, Faculty of Health and Medical Sciences, University of Copenhagen, Frederiksberg, Denmark.

The immunological mechanisms associated with protection of vaccinated rainbow trout, Oncorhynchus mykiss, against enteric redmouth disease (ERM), caused by Yersinia ruckeri, were previously elucidated by the use of gene expression methodology and immunochemical methods. That approach pointed indirectly to both humoral and cellular elements being involved in protection. The present study correlates the level of protection in rainbow trout to cellular reactions in spleen and head kidney and visualizes the processes by applying histopathological, immunohistochemical, and in situ hybridization techniques. It was shown that these cellular reactions, which were more prominent in spleen than in head kidney, were associated with the expression of immune-related genes, suggesting a Th2-like response. Y. ruckeri, as shown by in situ hybridization (ISH), was eliminated within a few days in vaccinated fish, whereas nonprotected fish still harbored bacteria for a week after infection. Vaccinated fish reestablished normal organ structure within a few days, whereas nonprotected fish showed abnormalities up to 1 month postinfection. Protection in the early phase of infection was mainly associated with the expression of genes encoding innate factors (complement factors, lysozyme, and acute phase proteins), but in the later phase of infection, increased expression of adaptive immune genes dominated. The histological approach used has shown that the cellular changes correlated with protection of vaccinated fish. They comprised transformation of resident cells into macrophage-like cells and increased occurrence of CD8α and IgM cells, suggesting these cells as main players in protection. Future studies should investigate the causality between these factors and protection.
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http://dx.doi.org/10.1128/CVI.00404-13DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3807201PMC
October 2013
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