Fast and accurate identification by MALDI-TOF of the zoonotic serovar E of Vibrio vulnificus linked to eel culture.

Vibrio vulnificus is a zoonotic pathogen that can cause death by septicaemia in farmed fish (mainly eels) and humans. The zoonotic strains that have been isolated from diseased eels and humans after eel handling belong to clade E (or serovar E (SerE)), a clonal complex within the pathovar (pv.) piscis. The aim of this study was to evaluate the accuracy of MALDI-TOF mass spectrometry (MS) in the identification of SerE, using the other two main pv. piscis-serovars (SerA and SerI) from eels as controls. MALDI-TOF data were compared with known serologic and genetic data of five pv. piscis isolates or strains, and with the non pv. piscis reference strain. Based on multiple spectra analysis, we found serovar-specific peaks that were of ~3098 Da and ~ 4045 Da for SerE, of ~3085 Da and ~ 4037 Da for SerA, and of ~3085 Da and ~ 4044 Da for SerI. Therefore, our results demonstrate that MALDI-TOF can be used to identify SerE and could also help in the identification of the other serovars of the species. This means that zoonosis due to V. vulnificus could be prevented by using MALDI-TOF, as action can be taken immediately after the isolation of a possible zoonotic V. vulnificus strain.

Reversible mutations in gliding motility and virulence genes: A flexible and efficient phage defence mechanism in Flavobacterium psychrophilum.

Flavobacteria are among the most important pathogens in freshwater salmonid aquaculture worldwide. Due to concerns regarding development of antibiotic resistance, phage therapy has been proposed as a solution to decrease pathogen load. However, application of phages is challenged by the development of phage resistance, and knowledge of the mechanisms and implications of phage resistance is therefore required. To study this, 27 phage-resistant isolates of F. psychrophilum were genome sequenced and characterized to identify genetic modifications and evaluate changes in phenotypic traits, including virulence against rainbow trout. Phage-resistant isolates showed reduction or loss of gliding motility, proteolytic activity, and adhesion to surfaces, and most isolates were completely non-virulent against rainbow trout fry. Genomic analysis revealed that most phage-resistant isolates had mutations in genes associated with gliding motility and virulence. Reversal of these mutations in a sub-set of isolates led to regained motility, proteolytic activity, virulence and phage susceptibility. Although costly, the fast generation of phage resistance driven by single, reversible mutations likely represents a flexible and efficient phage defence mechanism in F. psychrophilum. The results further suggest that phage administration in aquaculture systems to prevent F. psychrophilum outbreaks selects for non-virulent phage-resistant phenotypes.

Antigenic variability of Vibrio anguillarum serotype O2a: A hurdle for vaccine efficacy against vibriosis in rainbow trout (Oncorhynchus mykiss).

Despite vaccination, outbreaks of vibriosis still occur in sea-reared rainbow trout in Denmark. Vibriosis outbreaks are caused mainly by V. anguillarum serotypes O1 and O2a, and bacterins of both serotypes are included in the commonly used vaccine against this disease in Danish aquaculture. However, while the strains belonging to serotype O1 are genetically similar, the strains belonging to serotype O2a are highly diverse. This work aimed first at examining how the antibody response and protection induced by bacterin-based vaccines were affected by the antigenic variability within V. anguillarum serotype O2a strains. Following vaccination of rainbow trout with either a commercial or an experimental vaccine, specific antibody reactivity in serum from vaccinated fish was examined by ELISA against 23 strains of V. anguillarum serotype O2a (VaO2a). The strains were divided into 4 distinct subgroups according to the observed detection pattern. Seven strains were strongly recognized only by sera from fish vaccinated with the experimental vaccine (EV-I antisera), while 13 other strains were primarily recognized by sera from fish vaccinated with the commercial vaccine (CV antisera). Two strains were recognized by both EV-I and CV antisera, but with intermediate reactivity, while one strain was not recognized at all. A partly similar recognition pattern was observed when purified lipopolysaccharide (LPS) was used as antigen in the examination of antibody reactivity in Western blotting. The level of protection was highly dependent on both the vaccine and the strain used for challenge and showed no consistent correlation with antibody reactivity. Secondly, we attempted to use a bacterin vaccine based on one of the V. anguillarum O2a strains intermediately recognized by both EV-I and CV antisera to investigate whether that could potentially provide protection across strain variability. The immunized fish did mount a cross-reactive antibody response, but protection still varied depending on the strain used for challenge. Interestingly, the grouping of strains according to antibody reactivity correlated not only with genotyping based on single nucleotides polymorphisms analysis (SNP) but also with variability in the accessory genome, indicating that presence or absence of protein antigens or proteins associated with the biosynthesis of antigenic epitopes may explain the observed distinct serological subgrouping within VaO2a strains by trout immune sera. In terms of vaccination against VaO2a, our results demonstrate that it is important to take (local) antigen variations into account when using bacterin-based vaccines but also that alternatives to traditional bacterin-based vaccines might be needed to induce protection against the highly virulent Vibrio anguillarum serotype O2a strains.

Immersion vaccines against Yersinia ruckeri infection in rainbow trout: Comparative effects of strain differences.

The protective effects of autogenous and commercial ERM immersion vaccines (bacterins based on Yersinia ruckeri, serotype O1, biotypes 1 and 2) for rainbow trout (Oncorhynchus mykiss) were compared in order to evaluate whether the use of local pathogen strains for immunization can improve protection. In addition, the effect of the bacterin concentration was established for the commercial product. Following sublethal challenge of vaccinated and non-vaccinated control fish with live bacteria, we followed the bacterial count in the fish (gills, liver and spleen). The expression of genes encoding immune factors (IL-1ß, IL-6, IL-8, IL-10, IFN-γ, MHCI, MHCII, CD4, CD8, TCRß, IgM, IgT, IgD, cathelicidins 1 and 2, SAA and C3) and densities of immune cells in organs were recorded. Both vaccines conferred protection as judged from the reduced bacterial load in exposed fish. Innate immune genes were upregulated in all groups following bacterial challenge but significantly more in non-vaccinated naive fish in which densities of SAA-positive immune cells increased. Immunoglobulin genes were upregulated on day 5 post-challenge, and fish vaccinated with the high commercial bacterin dosage showed increased IgM levels by ELISA on day 14 post-challenge, reflecting that the vaccine dosage was correlated to protection. In conclusion, both vaccine types offered protection to rainbow trout when exposed to live Y. ruckeri and no significant difference between commercial and autogenous vaccines was established.

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

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 × 105 CFU mL-1) 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-1ß, 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-ß 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.

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

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.

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

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.

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

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.

A pentavalent vaccine for rainbow trout in Danish aquaculture.

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-1ß, IL-6, IL-8, IgD and MHCII.

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

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.

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

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.

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

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.

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

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.

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

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.

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

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

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.

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.

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

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-1ß 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.

Dispersal and survival of Flavobacterium psychrophilum phages in vivo in rainbow trout and in vitro under laboratory conditions: implications for their use in phage therapy.

Attention has been drawn to phage therapy as an alternative approach for controlling pathogenic bacteria such as Flavobacterium psychrophilum in salmonid aquaculture, which can give rise to high mortalities, especially in rainbow trout fry. Recently, phages have been isolated with a broad host range and a strong lytic potential against pathogenic F. psychrophilum under experimental conditions. However, little is known about the fate of phages at environmental conditions. Here, we quantified the dispersal and fate of F. psychrophilum phages and hosts in rainbow trout fry after intraperitoneal injection. Both phages and bacteria were isolated from the fish organs for up to 10 days after injection, and coinjection with both bacteria and phages resulted in a longer persistence of the phage in the fish organs, than when the fish had been injected with the phages only. The occurrence of both phage and bacterium was most prevalent in the kidney and spleen, with only minor occurrence in the brain. The experiment showed that injected phages were rapidly spread in the internal organs of the fish, also in the absence of bacteria. Parallel examination of the regulation of bacteriophage infectivity in controlled laboratory experiments at various environmental conditions showed that pH had only minor effects on long-term (3 months) phage infectivity within a pH range of 4.5 to 7.5, whereas phage infectivity was immediately lost at pH 3. In the absence of host cells, phage infectivity decreased by a factor of 10,000 over 55 days in untreated pond water, while the sterilization and removal of particles caused a 100-fold increase in phage survival relative to the control. In addition, F. psychrophilum-specific phages maintained their infectivity for ∼2 months in glycerol at -80°C, whereas infectivity decreased by a factor 10 when kept in a buffer at 20°C. Only a very small degradation in infectivity was seen when bacteriophages were added and dried on fish feed pellets. Together, these results indicate that application of bacteriophages represents a promising approach for the control of F. psychrophilum infections in trout and suggest fish feed as a potential delivery method.

Tissue specific uptake of inactivated and live Yersinia ruckeri in rainbow trout (Oncorhynchus mykiss): visualization by immunohistochemistry and in situ hybridization.

Understanding of uptake and invasion routes of Yersinia ruckeri, causing Enteric Red Mouth Disease (ERM) in rainbow trout (Oncorhynchus mykiss), is essential for improved understanding of the pathogenicity and immune response mechanisms associated this disease. The present work shed light on areas of invasion in rainbow trout by the use of immunohistochemistry and in situ hybridization techniques. Fish were exposed to live or formalin inactivated bacteria and samples were subsequently taken for histology from various outer and inner surfaces. We applied a specific monoclonal antibody and specific oligonucleotide probes binding to Y. ruckeri (serotype O1, biotype 2) in tissue sections and were able to demonstrate a tissue specific uptake of this bacterium (both formalin inactivated and live form). Uptake and subsequent translocation dynamics at various surfaces demonstrated different site specific propensities between the formalin inactivated and live bacterial organisms. Lateral lines, dorsal fin, epidermis and gastro-intestinal tract mucosal tissue were the primary areas where bacterial uptake was demonstrated readily after exposure. The fate of internalized bacterial organisms within the host suggested that central immune organs are involved in the final antigen processing.