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The development of effective vaccines is a critical step towards the domestication of emerging fish species for aquaculture. However, traditional vaccine delivery through intraperitoneal (i.p.) injection requires fish to reach a minimum size and age and therefore cannot provide protection at early developmental stages when infection may occur. This study investigated the effectiveness of immersion vaccination with respect to immunocompetence in a cleaner fish species (ballan wrasse, Labrus bergylta, Ascanius) used in Atlantic salmon farming as an alternative means to control sea lice. The species is susceptible to atypical strains of Aeromonas salmonicida (aAs) at early life stages (15 g), when i.p. vaccination is not applicable. While immersion vaccination is currently used in commercial hatcheries, the optimal fish size for vaccination, and efficacy of the vaccine delivered by this route has not yet been established. Importantly, efficacy depends on the capability of the species immune system to recognise antigens and process antigens to trigger and produce an adaptive immune response, (process known as immunocompetence). In this study, the efficacy of a polyvalent autogenous vaccine administered by immersion in juvenile ballan wrasse and the subsequent immune response induced was investigated after prime and booster vaccination regimes. In addition, temporal expression (0-150 days post hatch) of adaptive immune genes including major histocompatibility complex (MHC II CD74 molecule) and immunoglobulin M (IgM) was assessed using quantitative PCR (qPCR). Prime and/or boost vaccination by immersion of juvenile ballan wrasse (0.5 g and 1.5 g corresponding to 80 and 170 days post hatch (dph), respectively) did not provide significant protection against aAs vapA V after bath challenge under experimental conditions. Despite no evident protection80 dph, MHC II and IgM transcripts were first reported at 35 and 75 dph, respectively, suggesting a window of immunocompetence. The results provide important new information on the onset of adaptive immunity in ballan wrasse and highlight that immersion vaccination in the species for protection against aAs should be performed at later developmental stages (1.5 g) in the hatchery.

Flavobacterium psychrophilumis one of the most important bacterial pathogens affecting cultured rainbow trout (Oncorhynchus mykiss) and is increasingly causing problems in Atlantic salmon (Salmo salarL.) hatcheries. Little is known about the heterogeneity of F. psychrophilumisolates on UK salmonid farms. A total of 315 F. psychrophilumisolates, 293 of which were collected from 27 sites within the UK, were characterised using four genotyping methods and a serotyping scheme. A high strain diversity was identified among the isolates with 54 pulsotypes, ten (GTG)5-PCR types, two 16S rRNA allele lineages, seven plasmid profiles and three serotypes. Seven PFGE groups and 27 singletons were formed at a band similarity of 80%. PFGE group P (n=75) was found to be numerically predominant in eight sites within the UK. Two major PFGE clusters and 13 outliers were found at the band similarity of 40%. The predominant profile observed within the F. psychrophilumisolates examined was PFGE cluster II − (GTG)5-PCR type r1–16S rRNA lineage II − serotype Th (70/156 isolates examined, 45%). Co-existence of genetically and serologically heterogeneous isolates within each farm was detected, confounding the ability to control RTFS outbreaks. The occurrence over time (up to 11 years) of F. psychrophilumpulsotypes in three representative sites (Scot I, Scot III and Scot V) within Scotland was examined, potentially providing important epidemiological data for farm management and the development of site-specific vaccines.

Red mark syndrome (RMS) is a skin condition in Rainbow trout Oncorhynchus mykiss that has been reported worldwide but was first seen in the United Kingdom (UK) in 2003. The current study was conducted to examine if there was an association between a Midichloria-like organism (MLO) and RMS using a statistically appropriate sample set, whilst determining if there is a lack of association with Flavobacterium psychrophilum implicated in disease in previous studies. Fish in this study were obtained from three sites positive for RMS in the UK and United States (US), and three sites in the UK and the Netherlands that had no previous history of this condition. Samples taken from RMS-affected sites were found to show typical RMS pathology. Analysis of the major organs of affected fish by quantitative polymerase chain reaction (qPCR) demonstrated a significantly higher presence of the MLO in the RMS-affected tissues. Although most of the tissues were positive for the MLO, the highest correlation was seen in the skin, whilst the tissues from the unaffected fish were all negative. Thus, a strong positive correlation was found between the MLO and RMS-affected fish, whilst no association was found between the RMS-affected fish and F. psychrophilum other than superficial presence in the skin. The use of immunohistochemistry showed positive staining of what was considered to be MLO-related antigens in the internal organs of most RMS-affected fish. Attempts were made to culture the MLO, but no MLO was isolated.

Normalised resistance interpretive (NRI) analysis was applied to the development of interpretive criteria for antibiotic disc diffusion zones of mesophilic Aeromonas spp. Minimum quantitative and qualitative properties of data sets from which normalised resistance interpretation could generate acceptable cut-off values were established by examining published data that had been used to set the epidemiological cut-off values for bacteria of human and aquatic interest. Applying these criteria to disc diffusion data generated in a previous study of 129 mesophilic Aeromonas spp. isolated from pet fish, demonstrated that normalised resistance interpretation was capable of generating acceptable cut-off values for seven (erythromycin ≥ 8 mm, gentamicin ≥ 18 mm, moxalactam ≥ 32 mm, oxytetracycline ≥ 21 mm, streptomycin, ≥ 20 mm tetracycline ≥ 28 mm, and trimethoprim/sulfamethoxazole ≥ 26 mm) of the agents tested. This analysis demonstrated, with respect to these agents, that a single set of interpretive criteria could be developed for application to data generated from all the Aeromonads studied. Provisional cut-off values were generated for three other agents (chloramphenicol ≥ 35 mm, florfenicol ≥ 35 mm and furazolidone ≥ 23 mm). The distribution of zone sizes for these agents showed an apparent bimodality in the high-zone end. As a result, the question of whether, for these three agents, a single set of interpretive criteria could be validly set for all Aeromonads could not be resolved. With respect to the five quinolone agents studied, a cut-off value could only be estimated for oxolinic acid. Because of the high frequency of resistance to quinolones in the strain set, this value (≥ 32 mm) should be treated as a provisional estimate. With respect to some agents, strains manifesting a low-level resistant phenotype, zones ≤ 8 mm smaller than the wild-type cut-off, represented a significant proportion of the strains classified as non wild-type. The difficulties that these frequencies of low-level resistance would present for attempts to set and apply universal, laboratory-independent interpretive criteria are discussed.

Bacteria showing antimicrobial resistance (AMR) pose a significant global healthcare problem. Although many mechanisms conferring AMR are understood, the ecological processes facilitating its persistence and spread are less well characterised. Aquatic systems represent an important milieu for the environmental release, mixing, persistence and spread of AMR bacteria and resistance genes associated with horizontally transferable genetic elements. Additionally, owing to the use and discharge of antimicrobials and biocides, and the accumulation and abundance of other pollutants, mechanisms that confer AMR might evolve in aquatic systems. In this review, we hypothesise that aquatic systems have an important ecological and evolutionary role in driving the persistence, emergence and spread of AMR, which could have consequences when attempting to reduce its occurrence in clinical settings.

Number of enteric redmouth disease (ERM) outbreaks, caused by the bacterial fish pathogen Yersinia ruckeri, has increased considerably in Finnish fish farms since 2004. To evaluate if the Y. ruckeri isolates from the recent severe Finnish outbreaks represent a dominant strain and if they differ from older isolates, 44 Finnish isolates from 1986 to 2007 were characterised by biochemical tests, serotyping, pulsed-field gel electrophoresis (PFGE) and outer membrane protein analysis. The Finnish isolates were also compared with isolates from nearby geographical regions (Denmark and Sweden) and with the reference strain NCTC 10476. The results showed that most of the new isolates (2005–2007) from the Archipelago Sea (northern Baltic Sea) in Southwest Finland belong to biotype 2, which had not been previously reported from Finland. PFGE analysis indicated that the new biotype 2 isolates were very similar to isolates from Denmark (pulsotype 35) and that they most likely do not originate from the biotype 1 isolates obtained from fresh water hatcheries in Finland. Our conclusion is that the Y. ruckeri biotype 2 strain was introduced to Finnish fish farms in the Archipelago Sea from elsewhere.

A PCR protocol (McIntosh et al., 1996. Appl. Environ. Microbiol. 62, 3929–2932), designed to detect the P57 gene of Renibacterium salmoninarum , causative agent of Bacterial Kidney Disease, was modified slightly by redesign of the forward primer and recommended conditions to prevent possible false positives observed when tested against pure cultures of Yersinia ruckeri . The modified PCR, in combination with an improved DNAzol ™ -based DNA extraction technique, was very specific and sensitive (detecting between 5 and 72 R. salmoninarum cfu per mg head kidney tissue). It could detect infected fish using only 50 mg head kidney material, making the technique suitable for sampling small fish. It was also shown that samples could be pooled from up to five rainbow trout prior to PCR without noticeably affecting the sensitivity of the assay, providing at least 50 mg head kidney tissue was included from each of the fish that were pooled.

The potential of bacteriophage therapy to control bacterial disease in farmed fish was tested using, as an example, furunculosis of Atlantic salmon, caused by Aeromonas salmonicida subsp. salmonicida . In vivo testing with Atlantic salmon and rainbow trout ( Oncorhynchus mykiss Walbaum ) showed no adverse effects, with bacteriophage generally cleared within 96 h of administration by either intraperitoneal (i.p.) injection or oral in-feed. Juvenile Atlantic salmon were administered a combination of bacteriophage O, R and B (1.9 × 10 8 pfu fish − 1 ) by i.p. injection, after they had been challenged with A. salmonicida subsp. salmonicida 78027, also by i.p. injection. The fish that were injected with bacteriophage immediately after challenge died at a significantly slower rate then those that were either not treated with bacteriophage, or treated 24 h post-challenge. However, the end result (100% mortality) was not affected. In further experiments the effects of oral (1.88 × 10 5 pfu g − 1 fish − 1 daily for 30 days), bath (1.04 × 10 5 ml − 1 daily for 30 days) and i.p. (6.25 × 10 7 pfu fish − 1 ) phage treatment to control furunculosis in experimentally infected Atlantic salmon were compared with antibiotherapy (treatment with 10 mg kg − 1 bw − 1 day − 1 oxolinic acid for 10 days), using an indirect cohabitation challenge. No protection was offered by any of the bacteriophage treatments, compared to the positive challenge group, although significant protection was offered by the oxolinic acid treatment. Analysis of samples taken from the trials demonstrated that bacteriophage were correctly administered to the fish and, on occasion, were isolated from fish that had succumbed to furunculosis. It was also shown that bacteriophage resistant A. salmonicida subsp. salmonicida isolates could be recovered from mortalities in all the treatment groups. The results suggest that, although there were no safety problems associated with the approach, furunculosis in Atlantic salmon is not readily controllable by application of bacteriophage.

The microflora of rinsed eggs of Pacific threadfin, Polydactylus sexfilis, and amberjack, Seriola rivoliana, were characterised, using a combination of culture based and non-culture based methods. The adherent microflora of eggs, examined within a few hours of release, varied both qualitatively and quantitatively. Similar Pseudoalteromonas strains were recovered from the eggs of both species and Photobacterium damselae subsp. damselae was recovered from more than one batch of Pacific threadfin eggs. Many of the 16S rRNA gene sequences directly amplified and cloned from egg homogenate samples shared high homology to the partial 16S rRNA gene sequences of organisms cultured from the same samples of homogenate, suggesting that the majority of the bacteria in these samples were culturable on Marine Agar (MA). One of the organisms isolated from the eggs of Pacific threadfin, Vibrio str ME2-03, was shown to be highly virulent to hatched Pacific threadfin larvae in challenge experiments. This shows that, as with other fish species, egg-associated transfer of bacterial pathogens could be significant in Pacific threadfin culture, illustrating the likely importance of treating eggs prior to transport, either to other rearing areas or to other hatcheries.

The survival of Atlantic halibut ( Hippoglossus hippoglossus ) yolk-sac larvae and the development of their gut bacterial flora were compared in pilot-commercial scale rearing trials under different conditions. Four different treatments were tested; water recirculation using non-disinfected embryos (RN); water recirculation using disinfected embryos (RS); flow-to-waste water with disinfected embryos (F); flow-to-waste water with disinfected embryos and addition of antibiotics, oxytetracycline, oxolinic acid and amoxycillin at 20 mg l −1 (FA). Mean survival was greatest in the FA group (53%), which was significantly higher ( p 71% morphologically normal larvae in all treatments. Addition of antibiotics appeared to prevent the colonisation of yolk-sac larvae by Marine Agar (MA)-culturable bacteria ( 2 CFU/larva for group F). Higher levels of bacteria were recovered from both groups of larvae reared in recycled water (5.8×10 3 CFU/larva for group RN and 7.79×10 3 for group RS), indicating that bacterial abundance is not, in itself, a determinant of survival. Differences in the types of bacteria that colonised the halibut yolk-sac larvae under the different rearing regimes were described by characterization of the isolates. There appeared to be a relationship between larval survival rate and the presence, or absence, of certain phenotypes. In particular, the gut microflora in the poorest performing group (group F) was dominated by isolates biochemically and morphologically distinct from the predominant Pseudoalteromonas species isolated from the better performing recycled water groups. This lends weight to the hypothesis that certain types of bacteria may act as opportunistic pathogens in yolk-sac incubators while others are benign.

The bacterial flora of reared Atlantic halibut eggs, larvae, juveniles and adults from three different UK halibut hatcheries was monitored using a combination of traditional biochemical tests, the BIOLOG GN bacterial identification system, PCR-RFLP of 16S rRNA genes and partial 16S rDNA gene analysis. A gut microflora was detected towards the beginning of the non-feeding yolk-sac stage, with a substantial increase as the larvae started to feed. Bacteria isolated from non-feeding yolk-sac larvae were predominantly non-fermentative Gram-negative rods, in particular Pseudoalteromonas species; by contrast, the presumptive gut microflora of first-feeding larvae was generally dominated by members of the Vibrio genus. There was evidence of a bacterial succession after the larvae started to feed. Initially, first-feeding halibut reared on enriched Artemia were generally colonised by live food-associated bacteria, particularly Vibrio splendidus and V. alginolyticus-type organisms. Genetically similar V. splendidus isolates were recovered from enriched Artemia and the guts of first-feeding halibut larvae in all three hatcheries. However, only a selected proportion of the Artemia-associated bacteria appeared able to establish themselves within the guts of larval halibut. First-feeding halibut larvae were sometimes colonised by opportunistic bacteria. The gut floras of two individual batches of halibut larvae were briefly dominated by a Pseudoalteromonas and a Photobacterium phosphoreum isolate, respectively. Genetically-similar V. salmonicida-type organisms were present in larvae sampled from all three hatcheries, both from copepod and Artemia-fed fish. The guts of the adult halibut analysed had a culturable flora dominated by Photo. phosphoreum.

Experimental bath challenge of juvenile and sub-adult Atlantic halibut with Vibrio anguillarum induced severe mortalities of 47 and 80%, respectively. However, animals vaccinated with a commercial V. anguillarum vaccine demonstrated excellent protection against the disease (100% RPS). This study also describes the gross pathology and histological changes associated with this infection. A loss of coordination, haemorrhage at the fin base and splenomegaly were frequent findings. Serum agglutinating activity demonstrated a rise following vaccination, the mean log2 titre rising from 3.8 to 8.4. This was associated with a significant rise in antibody-mediated complement killing ability of immune serum when compared to non-immune serum.