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2.AbstractBacteria from the Vibrionaceae family have been implicated in mass mortalities of farmed Pacific oysters (Crassostrea gigas) in multiple countries, leading to substantial impairment for growth in the sector. In Ireland there has been concern thatVibriohave been involved in serious summer outbreaks. There is evidence thatVibrio aestuarianusis increasingly becoming the main pathogen of concern for the Pacific Oyster industry in Ireland. While bacteria belonging to theVibrio splendidusclade are also detected frequently in mortality episodes, their role in the outbreaks of summer mortality are not well understood. To identify and characterise strains involved in these outbreaks, 43Vibrioisolates were recovered from Pacific oyster summer mass mortality episodes in Ireland from 2008-2015 and these were whole genome sequenced. Among these, 25 were found to beV. aestuarianus(implicated in disease) and 18V. splendidus sensu lato(role in disease undetermined). Two distinct clades ofV. aestuarianus– Clade A and Clade B – were found that had previously been described as circulating within French oyster culture. The high degree of similarity between the Irish and FrenchV. aestuarianusisolates points to translocation of the pathogen between Europe’s two major oyster producing countries, probably via trade in spat and other age classes.V. splendidusisolates were more diverse, but the data reveal a single clone of this species that has spread across oyster farms in Ireland. This underscores thatVibriocould be transmitted readily across oyster farms. The presence ofV. aestuarianusClades A and B in not only France but also Ireland adds weight to growing concern that this pathogen is spreading and impacting Pacific oyster production within Europe.3.OutcomePacific oyster culture in Ireland has increasingly suffered from summer mass mortality events. Many of these mortalities in recent years have been associated withVibrio aestuarianus; the role of another pathogen,Vibrio splendidushas, so far, remained inconclusive. Here we show that two clades ofV. aestuarianusare circulating in Ireland, and that these are members of two clades that have previously caused extensive oyster die offs in France. Their discovery in Ireland is consistent with transport of infected oyster stock between the two countries. AlthoughV. splendidus-like strains in Ireland were highly diverse, a small clonal group was detected that appears to have spread rapidly from a single source to disparate locations in Ireland. Combined, these findings highlight the appearance of a highly pathogenicVibrioin Ireland, and the risk of transmission between interconnected oyster production industries in Europe.4.Data summarySequences generated in this study were deposited on the NCBI. Accession number: PRJNA797364. Publicly accessed genomes are listed in Table S2.The authors confirm all supporting data, code and protocols have been provided within the article or through supplementary data files.

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.

While both virulent and putatively avirulent Yersinia ruckeri strains exist in aquaculture environments, the relationship between the distribution of virulence-associated factors and de facto pathogenicity in fish remains poorly understood. Pan-genome analysis of 18 complete genomes, representing established virulent and putatively avirulent lineages of Y. ruckeri, revealed the presence of a number of accessory genetic determinants. Further investigation of 68 draft genome assemblies revealed that the distribution of certain putative virulence factors correlated well with virulence and host-specificity. The inverse-autotransporter invasin locus yrIlm was, however, the only gene present in all virulent strains, while absent in lineages regarded as avirulent. Strains known to be associated with significant mortalities in salmonid aquaculture display a combination of serotype O1-LPS and yrIlm, with the well-documented highly virulent lineages, represented by MLVA clonal complexes 1 and 2, displaying duplication of the yrIlm locus. Duplication of the yrIlm locus was further found to have evolved over time in clonal complex 1, where some modern, highly virulent isolates display up to three copies.

Genetic resistance to infectious pancreatic necrosis virus (IPNV) in Atlantic salmon is a rare example of a trait where a single locus (QTL) explains almost all of the genetic variation. Genetic marker tests based on this QTL on salmon chromosome 26 have been widely applied in selective breeding to markedly reduce the incidence of the disease. In the current study, whole genome sequencing and functional annotation approaches were applied to characterise genes and variants in the QTL region. This was complemented by an analysis of differential expression between salmon fry of homozygous resistant and homozygous susceptible genotypes challenged with IPNV. These analyses pointed to the NEDD-8 activating enzyme 1 (nae1) gene as a putative functional candidate underlying the QTL effect. The role of nae1 in IPN resistance was further assessed via CRISPR-Cas9 knockout of the nae1 gene and chemical inhibition of the nae1 protein activity in Atlantic salmon cell lines, both of which resulted in highly significant reduction in productive IPNV replication. In contrast, CRISPR-Cas9 knockout of a candidate gene previously purported to be a cellular receptor for the virus (cdh1) did not have a major impact on productive IPNV replication. These results suggest that nae1 is the causative gene underlying the major QTL affecting resistance to IPNV in salmon, provide further evidence for the critical role of neddylation in host-pathogen interactions, and highlight the value in combining high-throughput genomics approaches with targeted genome editing to understand the genetic basis of disease resistance., Highlights • Whole genome sequencing and functional annotation were used to characterise the major IPNV resistance QTL in Atlantic salmon. • Gene expression analyses pointed to NEDD-8 activating enzyme 1 (nae1) as a putative functional candidate underlying the QTL. • CRISPR-Cas9 knockout and chemical inhibition of nae1 in cell lines resulted in significant reduction in IPNV replication. • CRISPR-Cas9 knockout of a previously suggested candidate gene (cdh1) did not have an impact on productive IPNV replication. • These results suggest that nae1 is the causative gene underlying the major QTL affecting resistance to IPNV in salmon.

Improved biosecurity and livestock disease control measures in low resource settings are often regarded as beneficial for agricultural productivity, rural incomes, global health, and sustainability. In this paper we present data from a study of shrimp and prawn aquaculture in Bangladesh to argue that this relationship is not as straightforward as it would seem. Analysing quantitative and qualitative data from a multi‐method field study involving 300 “missing middle” farmers, we demonstrate the importance of socio‐economic and ecological conditions to any disease management strategy. We describe how a technical programme to introduce “disease‐free” seed faltered partly as a result of the farmers' tendency to offset disease and livelihood risks by frequently re‐stocking their ponds. Changes to seed provision were accompanied by calls to alter farmers' livestock production practices. Paradoxically, these changes exposed farmers to more intense risks, potentially locking them into unsustainable disease management practices. The analysis emphasises that vernacular farming practices should be considered as key assets rather than barriers to disease management strategies, and that closer attention be paid to value chain and other risks as drivers of unsustainable practices.

Aquaculture is predicted to supply the majority of aquatic dietary protein by 2050. For aquaculture to deliver significantly enhanced volumes of food in a sustainable manner, appropriate account needs to be taken of its impacts on environmental integrity, farmed organism health and welfare, and human health. Here, we explore increased aquaculture production through the One Health lens and define a set of success metrics — underpinned by evidence, policy and legislation — that must be embedded into aquaculture sustainability. We provide a framework for defining, monitoring and averting potential negative impacts of enhanced production — and consider interactions with land-based food systems. These metrics will inform national and international science and policy strategies to support improved aquatic food system design. By 2050, the majority of aquatic dietary protein will be produced by the aquaculture sector. A set of 15 metrics are presented here to guide the industry sustainably through the rapid growth and development it is experiencing.

Atypical Aeromonas salmonicida (aAs) is currently one of the most routinely recovered bacterial pathogens isolated during disease outbreaks in farmed cleaner fish, ballan wrasse (Labrus bergylta, Ascanius). Vibrionaceae family bacteria have also been isolated from ballan wrasse in Scotland. This study determined the infectivity, pathogenicity and virulence of aAs and Vibrionaceae isolates in juvenile farmed ballan wrasse (n= 50; approx. 2 gr) using a bath challenge and fish monitored for a period of 16 days. Atypical As caused significant mortalities in contrast to Vibrionaceae isolates. Notably, differential virulence was observed between two aAs vapA type V strains at similar challenge doses. Diseased fish exhibited a systemic infection where aAs was detected in all analysed tissues (liver, spleen and kidney) by PCR and qPCR. Macroscopically, moribund and survivor fish exhibited hepatomegaly and splenomegaly. In moribund and surviving fish histopathology showed granulomatous hepatitis with eosinophilic granular cells surrounding bacterial colonies and endocarditis along with splenic histiocytosis. This is the first report of a successful aAs bath challenge model for juvenile ballan wrasse which provides an important tool for future studies on vaccine efficacy and immunocompetence.

Antimicrobial resistance (AMR) is a growing threat to global public health, and the overuse of antibiotics in animals has been identified as a major risk factor. With high levels of international trade and direct connectivity to the aquatic environment, shrimp aquaculture may play a role in global AMR dissemination. The vast majority of shrimp production occurs in low‐ and middle‐income countries, where antibiotic quality and usage is widely unregulated, and where the integration of aquaculture with family livelihoods offers many opportunities for human, animal and environmental bacteria to come into close contact. Furthermore, in shrimp growing areas, untreated waste is often directly eliminated into local water sources. These risks are very different to many other major internationally‐traded aquaculture commodities, such as salmon, which is produced in higher income countries where there are greater levels of regulation and well‐established management practices. Assessing the true scale of the risk of AMR dissemination in the shrimp industry is a considerable challenge, not least because obtaining reliable data on antibiotic usage is very difficult. Combating the risks associated with AMR dissemination is also challenging due to the increasing trend towards intensification and its associated disease burden, and because many farmers currently have no alternatives to antibiotics for preventing crop failure. In this review, we critically assess the potential risks the shrimp industry poses to AMR dissemination. We also discuss some of the possible risk mitigation strategies that could be considered by the shrimp industry as it strives for a more sustainable future in production.

Laboratory animals need to be monitored to check the status of their health and welfare. Routine checks of laboratory fish are limited to visual observations of physical appearance and behaviour, but for species held in opaque-walled tanks, such checks are compromised by restricted views, poor visibility and provoked behaviour. Here, we report our experience of using in-tank underwater cameras to monitor laboratory populations of salmoniforme, perciforme and cypriniforme fish. A range of cameras and lenses were investigated and trialled. A standard VGA resolution analogue camera with a one-third-inch chip and 3.6-mm lens was selected based on size, picture quality, the proportion of tank in view and cost. A shell for the camera and mounting system were designed to minimise size and cleaning and enable flexible positioning within tanks. Cameras were connected via digital encoders to a server, making video available to the general computer network. Data collected from recordings of rainbow trout (Oncorhynchus mykiss) confirmed provoked behaviour, ie a change in distribution and increase in activity in response to direct viewing and feeding. The networked cameras therefore enable remote viewing of undisturbed behaviour in real time, providing clear, lateral views unaffected by water surface effects, and facilitate increased frequency of checking. Case studies illustrate how camera monitoring can aid detection of abnormalities in behaviour (eg lack of feeding, posture, swimming) and appearance (eg clinical signs, such as lesions), enabling earlier interventions. Furthermore, recordings provide a resource for reference and retrospective analysis, and evidence to support severity classification and identify humane end-points.

Atypical Aeromonas salmonicida (aAs) and Vibrionaceae related species are bacteria routinely recovered from diseased ballan wrasse used as cleaner fish in the Atlantic salmon farming industry. Autogenous (i.e. farm specific inactivated) multivalent vaccines formulated from these microorganisms are widely used to protect farmed wrasse despite limited experimental proof that they are primary pathogens. In this study, the components of a commercial multivalent injection vaccine containing four strains of Aeromonas salmonicida and one strain of Vibrio splendidus previously isolated from ballan wrasse in Scotland, were tested for infectivity, pathogenicity and virulence via intra peritoneal injection at pre-deployment size (25–50 g) and the efficacy of the vaccine for protection against aAs assessed. Injection with 3.5 × 109, 8 × 109 1.8 × 109 and 5 × 109 cfu/fish of Vibrio splendidus, V. ichthyoenteri, Aliivibrio logeii and A. salmonicida, respectively, did not cause significant mortalities, lesions or clinical signs after a period of 14 days. IP injection with both aAs and Photobacterium indicum successfully reproduced the clinical signs and internal lesions observed during natural outbreaks of the disease. Differences in virulence (LD50 at day 8-post infection of 3.6 × 106 cfu/fish and 1.6 × 107 cfu/fish) were observed for two aAs vapA type V isolates. In addition, the LD50 for Photobacterium indicum was 2.2 × 107 cfu/fish. The autogenous vaccine was highly protective against the two aAs vapA type V isolates after 700-degree days of immunisation. The RPSFINAL values for the first isolate were 95 and 91% at 1 × 106 cfu/fish and 1 × 107 cfu/fish, respectively, and 79% at 1 × 107 cfu/fish for the second isolate tested. In addition, significantly higher anti aAs seral antibodies (IgM), were detected by ELISA in vaccinated fish in contrast with control (mock vaccinated) fish. These results suggest wrasse can be effectively immunised and protected against aAs infection by injection with oil adjuvanted vaccines prepared with inactivated homologous isolates.

Here, we report the complete genome of piscine Streptococcus agalactiae 01173 serotype Ia, which was generated using long-read sequencing technology. The bacteria were isolated from wild fish displaying signs of streptococcosis, from a fish kill incident in Kuwait.

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.

Wild-caught ballan wrasse Labrus bergylta are translocated en masse from the British south-west coast to Scotland for use as cleaner fish to tackle Atlantic salmon Salmo salar sea lice infestations; however, very little is known about the background health status of this species. This is the first health assessment of wild ballan wrasse from the British south-west. Wild-caught ballan wrasse (n = 75) from coastal populations off Dorset and Cornwall were subjected to a full health screen for viral, bacterial and parasitic infections and associated pathology. A range of metazoan and protozoan parasites were observed in histological sections, including copepods (sea lice Caligus centrodonti), nematodes, cestodes, digenean metacercariae, Cryptocaryon-like ciliates and an intestinal coccidian (Eimeria sp.) observed in 26.6% of the samples. The mycoplasma Acholeplasma laidlawii was associated with cytopathic effect in cell culture inoculated with tissue homogenates. The opportunistic pathogen Photobacterium damselae damselae was isolated from a single fish with a systemic infection. The isolate was confirmed to possess the virulence factors hlyAch and plpV, previously associated with cell toxicity and pathogenicity to fish. There are no immediate concerns for the continued mass translation of ballan wrasse, however careful monitoring of the population is recommended.

SummaryIn late 2018, unusual patterns of very high mortality (>50% production) were reported in intensive tilapia cage culture systems across Lake Volta in Ghana. Samples of fish and fry were collected and analysed from two affected farms between October 2018 and February 2019. Affected fish showed darkening, erratic swimming and abdominal distension with associated ascites. Histopathological observations of tissues taken from moribund fish at different farms revealed lesions indicative of viral infection. These included haematopoietic cell nuclear and cytoplasmic pleomorphism with marginalisation of chromatin and fine granulation. Transmission electron microscopy showed cells contained conspicuous virions with typical Iridovirus morphology i.e. enveloped, with icosahedral and or polyhedral geometries and with a diameter c.160 nm. PCR confirmation and DNA sequencing identified the virions as Infectious Spleen and Kidney Necrosis Virus (ISKNV). Samples of fry and older animals were all strongly positive for the presence of the virus by qPCR. All samples tested negative for TiLV and Nodavirus by qPCR. All samples collected from farms prior to the mortality event were negative for ISKNV. Follow up testing of fish and fry sampled from 5 additional sites in July 2019 showed all farms had fish that were PCR positive for ISKNV, whether there was active disease on the farm or not, demonstrating the disease was endemic to farms all over Lake Volta by that point. The results suggest that ISKNV was the cause of disease on the investigated farms and likely had a primary role in the mortality events. A common observation of coinfections with Streptococcus agalactiae and other tilapia bacterial pathogens further suggests that these may interact to cause severe pathology, particularly in larger fish. Results demonstrate that there are a range of potential threats to the sustainability of tilapia aquaculture that need to be guarded against.

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., Sub-lineages of the fish-pathogenic bacterium Aeromonas salmonicida display specific host preferences.

Aquaculture is the fastest growing food-producing sector, and the sustainability of this industry is critical both for global food security and economic welfare. The management of infectious disease represents a key challenge. Here, we discuss the opportunities afforded by whole genome sequencing of bacterial and viral pathogens of aquaculture to mitigate disease emergence and spread. We outline, by way of comparison, how sequencing technology is transforming the molecular epidemiology of pathogens of public health importance, emphasizing the importance of community-oriented databases and analysis tools.

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.

This comprehensive population study substantially improves our understanding of the epizootiological history and nature of an internationally important fish-pathogenic bacterium. The MLVA assay developed and presented represents a high-resolution typing tool particularly well suited for Yersinia ruckeri infection tracing, selection of strains for vaccine inclusion, and risk assessment. The ability of the assay to separate isolates into geographically linked and/or possibly host-specific clusters reflects its potential utility for maintenance of national biosecurity. The MLVA is internationally applicable and robust, and it provides clear, unambiguous, and easily interpreted results. Typing is reasonably inexpensive, with a moderate technological requirement, and may be completed from a harvested colony within a single working day. As the resulting MLVA profiles are readily portable, any Y. ruckeri strain may rapidly be placed in a global epizootiological context., A multilocus variable-number tandem-repeat analysis (MLVA) assay was developed for epizootiological study of the internationally significant fish pathogen Yersinia ruckeri, which causes yersiniosis in salmonids. The assay involves amplification of 10 variable-number tandem-repeat (VNTR) loci in two five-plex PCRs, followed by capillary electrophoresis. A collection of 484 Y. ruckeri isolates, originating from various biological sources and collected from four continents over 7 decades, was analyzed. Minimum-spanning-tree cluster analysis of MLVA profiles separated the studied population into nine major clonal complexes and a number of minor clusters and singletons. The major clonal complexes could be associated with host species, geographic origin, and serotype. A single large clonal complex of serotype O1 isolates dominating the yersiniosis situation in international rainbow trout farming suggests anthropogenic spread of this clone, possibly related to transport of fish. Moreover, subclustering within this clonal complex indicates putative transmission routes and multiple biotype shift events. In contrast to the situation in rainbow trout, Y. ruckeri strains associated with disease in Atlantic salmon appear as more or less geographically isolated clonal complexes. A single complex of serotype O1 exclusive to Norway was found to be responsible for almost all major yersiniosis outbreaks in modern Norwegian salmon farming, and site-specific subclustering further indicates persistent colonization of freshwater farms in Norway. Identification of genetically diverse Y. ruckeri isolates from clinically healthy fish and environmental sources also suggests the widespread existence of less-virulent or avirulent strains. IMPORTANCE This comprehensive population study substantially improves our understanding of the epizootiological history and nature of an internationally important fish-pathogenic bacterium. The MLVA assay developed and presented represents a high-resolution typing tool particularly well suited for Yersinia ruckeri infection tracing, selection of strains for vaccine inclusion, and risk assessment. The ability of the assay to separate isolates into geographically linked and/or possibly host-specific clusters reflects its potential utility for maintenance of national biosecurity. The MLVA is internationally applicable and robust, and it provides clear, unambiguous, and easily interpreted results. Typing is reasonably inexpensive, with a moderate technological requirement, and may be completed from a harvested colony within a single working day. As the resulting MLVA profiles are readily portable, any Y. ruckeri strain may rapidly be placed in a global epizootiological context.

Renibacterium salmoninarum is the causative agent of bacterial kidney disease (BKD), which is a commercially important disease of farmed salmonids. Typing by conventional methods provides limited information on the evolution and spread of this pathogen, as there is a low level of standing variation within the R. salmoninarum population. Here, we apply whole-genome sequencing to 42 R. salmoninarum isolates from Chile, primarily from salmon farms, in order to understand the epidemiology of BKD in this country. The patterns of genomic variation are consistent with multiple introductions to Chile, followed by rapid dissemination over a 30 year period. The estimated dates of introduction broadly coincide with major events in the development of the Chilean aquaculture industry. We find evidence for significant barriers to transmission of BKD in the Chilean salmon production chain that may also be explained by previously undescribed signals of host tropism in R. salmoninarum. Understanding the genomic epidemiology of BKD can inform disease intervention and improve sustainability of the economically important salmon industry. This article contains data hosted by Microreact.

Puffy skin disease (PSD) is an emerging skin condition which affects rainbow trout, Oncorhynchus mykiss (Walbaum). The transmission pattern of PSD suggests an infectious aetiology, however, the actual causative infectious agent(s) remain(s) unknown. In the present study, the rainbow trout epidermal immune response to PSD was characterised. Skin samples from infected fish were analysed and classified as mild, moderate or severe PSD by gross pathology and histological assessment. The level of expression of 26 immune-associated genes including cytokines, immunoglobulins and cell markers were examined by TaqMan qPCR assays. A significant up-regulation of the gene expression of C3, lysozyme, IL-1β and T-bet and down-regulation of TGFβ and TLR3 was observed in PSD fish compared to control fish. MHCI gene expression was up-regulated only in severe PSD lesions. Histological examinations of the epidermis showed a significant increase in the number of eosinophil cells and dendritic melanocytes in PSD fish. In severe lesions, mild diffuse lymphocyte infiltration was observed. IgT and CD8 positive cells were detected locally in the skin of PSD fish by in situ hybridisation (ISH), however, the gene expression of those genes was not different from control fish. Total IgM in serum of diseased animals was not different from control fish, measured by a sandwich ELISA, nor was significant up regulation of IgM gene expression in PSD lesions observed. Taken together, these results show activation of the complement pathway, up-regulation of a Th17 type response and eosinophilia during PSD. This is typical of a response to extracellular pathogens (i.e. bacteria and parasites) and allergens, commonly associated with acute dermatitis.

One of the fastest growing fisheries in the UK is the king scallop ( Pecten maximus L.), also currently rated as the second most valuable fishery. Mass mortality events in scallops have been reported worldwide, often with the causative agent(s) remaining uncharacterized. In May 2013 and 2014, two mass mortality events affecting king scallops were recorded in the Lyme Bay marine protected area (MPA) in Southwest England. Histopathological examination showed gill epithelial tissues infected with intracellular microcolonies (IMCs) of bacteria resembling Rickettsia -like organisms (RLOs), often with bacteria released in vascular spaces. Large colonies were associated with cellular and tissue disruption of the gills. Ultrastructural examination confirmed the intracellular location of these organisms in affected epithelial cells. The 16S rRNA gene sequences of the putative IMCs obtained from infected king scallop gill samples, collected from both mortality events, were identical and had a 99.4% identity to 16S rRNA gene sequences obtained from “ Candidatus Endonucleobacter bathymodioli” and 95% with Endozoicomonas species. In situ hybridization assays using 16S rRNA gene probes confirmed the presence of the sequenced IMC gene in the gill tissues. Additional DNA sequences of the bacterium were obtained using high-throughput (Illumina) sequencing, and bioinformatic analysis identified over 1,000 genes with high similarity to protein sequences from Endozoicomonas spp. (ranging from 77 to 87% identity). Specific PCR assays were developed and applied to screen for the presence of IMC 16S rRNA gene sequences in king scallop gill tissues collected at the Lyme Bay MPA during 2015 and 2016. There was 100% prevalence of the IMCs in these gill tissues, and the 16S rRNA gene sequences identified were identical to the sequence found during the previous mortality event. IMPORTANCE Molluscan mass mortalities associated with IMCs have been reported worldwide for many years; however, apart from histological and ultrastructural characterization, characterization of the etiological agents is limited. In the present work, we provide detailed molecular characterization of an Endozoicomonas -like organism (ELO) associated with an important commercial scallop species.

Routine application of antimicrobials is the current treatment of choice for rainbow trout fry syndrome (RTFS) or bacterial coldwater disease (BCWD) caused by Flavobacterium psychrophilum. In this study, the antimicrobial susceptibilities of 133 F. psychrophilum isolates, 118 of which were from the UK, were evaluated by broth microdilution and disc diffusion methods following VET04-A2 and VET03-A guidelines of Clinical and Laboratory Standards Institute (CLSI), respectively. Isolates were categorized as wild type (fully susceptible, WT) or non-wild type (NWT) using normalized resistance interpretation (NRI)-determined cut-off values (COWT ). Broth microdilution testing showed that only 12% of UK isolates were WT to oxolinic acid (MIC COWT ≤ 0.25 mg/L) and 42% were WT for oxytetracycline (MIC COWT ≤ 0.25 mg/L). In contrast, all the isolates tested were WT (MIC COWT ≤ 2 mg/L) for florfenicol, the main antimicrobial for RTFS control in the UK. Disc diffusion-based COWT values were ≥51 mm for 10 μg amoxicillin, ≥44 mm for 30 μg florfenicol, ≥30 mm for 2 μg oxolinic acid and ≥51 mm for 30 μg oxytetracycline. There was a high categorical agreement between the classifications of the isolates by two testing methods for florfenicol (100%), oxytetracycline (93%) and oxolinic acid (99%).

Bacteria from the family Flavobacteriaceae often show low susceptibility to antibiotics. With the exception of two Chryseobacteriumspp. isolates that were positive for the florfenicol resistance gene floR, no clinical resistance genes were identified by microarray in 36 Flavobacteriaceae isolates from salmonid fish that could grow in ≥ 4 mg/L florfenicol. Whole genome sequence analysis of the floRpositive isolates revealed the presence of a region that contained the antimicrobial resistance genes floR, a tet(X) tetracycline resistance gene, a streptothricin resistance gene and a chloramphenicol acetyltransferase gene. In silicoanalysis of 377 published genomes for Flavobacteriaceae isolates from a range of sources confirmed that well-characterised resistance gene cassettes were not widely distributed in bacteria from this group. Efflux pump-mediated decreased susceptibility to a range of antimicrobials was confirmed in both floRpositive isolates using an efflux pump inhibitor (phenylalanine-arginine β-naphthylamide) assay. The floRisolates possessed putative virulence factors, including production of siderophores and haemolysins, and were mildly pathogenic in rainbow trout. Results support the suggestion that, despite the detection of floR, susceptibility to antimicrobials in Flavobacteriaceae is mostly mediated via intrinsic mechanisms rather than the horizontally acquired resistance genes more normally associated with Gram-negative bacterial pathogens such as Enterobacteriaceae.

Fisheries and aquaculture production, imports, exports and equitability of distribution determine the supply of aquatic food to people. Aquatic food security is achieved when a food supply is sufficient, safe, sustainable, shockproof and sound: sufficient, to meet needs and preferences of people; safe, to provide nutritional benefit while posing minimal health risks; sustainable, to provide food now and for future generations; shock-proof, to provide resilience to shocks in production systems and supply chains; and sound, to meet legal and ethical standards for welfare of animals, people and environment. Here, we present an integrated assessment of these elements of the aquatic food system in the United Kingdom, a system linked to dynamic global networks of producers, processors and markets. Our assessment addresses sufficiency of supply from aquaculture, fisheries and trade; safety of supply given biological, chemical and radiation hazards; social, economic and environmental sustainability of production systems and supply chains; system resilience to social, economic and environmental shocks; welfare of fish, people and environment; and the authenticity of food. Conventionally, these aspects of the food system are not assessed collectively, so information supporting our assessment is widely dispersed. Our assessment reveals trade-offs and challenges in the food system that are easily overlooked in sectoral analyses of fisheries, aquaculture, health, medicine, human and fish welfare, safety and environment. We highlight potential benefits of an integrated, systematic and ongoing process to assess security of the aquatic food system and to predict impacts of social, economic and environmental change on food supply and demand.

Farmed and wild salmonids are affected by a variety of skin conditions, some of which have significant economic and welfare implications. In many cases, the causes are not well understood, and one example is cold water strawberry disease of rainbow trout, also called red mark syndrome, which has been recorded in the UK since 2003. To date, there are no internationally agreed methods for describing these conditions, which has caused confusion for farmers and health professionals, who are often unclear as to whether they are dealing with a new or a previously described condition. This has resulted, inevitably, in delays to both accurate diagnosis and effective treatment regimes. Here, we provide a standardized methodology for the description of skin conditions of rainbow trout of uncertain aetiology. We demonstrate how the approach can be used to develop case definitions, using coldwater strawberry disease as an example.

Yersinia ruckeri is the etiological agent of enteric redmouth (ERM) disease of farmed salmonids. Enteric redmouth disease is traditionally associated with rainbow trout ( Oncorhynchus mykiss , Walbaum), but its incidence in Atlantic salmon ( Salmo salar ) is increasing. Yersinia ruckeri isolates recovered from diseased Atlantic salmon have been poorly characterized, and very little is known about the relationship of the isolates associated with these two species. Phenotypic approaches were used to characterize 109 Y. ruckeri isolates recovered over a 14-year period from infected Atlantic salmon in Scotland; 26 isolates from infected rainbow trout were also characterized. Biotyping, serotyping, and comparison of outer membrane protein profiles identified 19 Y. ruckeri clones associated with Atlantic salmon but only five associated with rainbow trout; none of the Atlantic salmon clones occurred in rainbow trout and vice versa . These findings suggest that distinct subpopulations of Y. ruckeri are associated with each species. A new O serotype (designated O8) was identified in 56 biotype 1 Atlantic salmon isolates and was the most common serotype identified from 2006 to 2011 and in 2014, suggesting an increased prevalence during the time period sampled. Rainbow trout isolates were represented almost exclusively by the same biotype 2, serotype O1 clone that has been responsible for the majority of ERM outbreaks in this species within the United Kingdom since the 1980s. However, the identification of two biotype 2, serotype O8 isolates in rainbow trout suggests that vaccines containing serotypes O1 and O8 should be evaluated in both rainbow trout and Atlantic salmon for application in Scotland. IMPORTANCE Vaccination plays an important role in protecting Atlantic salmon against the bacterial pathogen Yersinia ruckeri , but, in recent years, there has been an increasing incidence of vaccine breakdown in salmon. This is largely because current vaccines are aimed at rainbow trout and are based on serotypes specific for this species. A wider range of serotypes is responsible for infection in Atlantic salmon, but very little is known about the diversity of these strains and their relationships to those recovered from rainbow trout. In the present study, we demonstrate that Y. ruckeri isolates recovered from diseased Atlantic salmon in Scotland are more diverse than those from rainbow trout; furthermore, isolates from the two species represent distinct subpopulations. In addition, a new O serotype was identified that is responsible for a significant proportion of the disease in Atlantic salmon. Our findings are likely to have important implications for the development of improved vaccines against Y. ruckeri .

The transmission of puffy skin disease (PSD) to rainbow trout Oncorhynchus mykiss Walbaum was tested in the laboratory by conducting co-habitation challenges with puffy skin (PS)-affected fish (Trojans) collected from the field. Two separate challenges were conducted using Trojans sourced from two different sites and diploid (first trial) or triploid (second trial) naïve fish. PSD-specific clinical signs were observed in both groups of naïve fish, with 66% of the fish sampled during the challenges showing signs of varying severity. The first clinical features of PSD were presented as white oval skin patches on one or both flanks 15-21 days post-challenge (dpc). The extent of the lesions ranged from 10 to 90% of the body surface, depending on the severity of the lesion. Both the severity and number of affected fish increased during the challenge. Macroscopically, oedema of the skin and multifocal petechial haemorrhaging were observed towards the end of the trials. Abnormal fish behaviour consisting of "flashing" and excessive mucous production was noted from 15 dpc onwards. Fish with severe PSD lesions also displayed inappetence and associated emaciation. Rodlet cells were observed in 41% of the fresh skin scrapes analysed from the second trial. Histologically epidermal oedema was observed in 31% of the naive fish showing gross pathology, with additional 12% displaying epidermal hyperplasia, mostly observed at the end of the challenge. Other concomitant features of the PSD lesions in challenged fish were epithelial erosion and sloughing, and occasionally mild or focal inflammation. No consistent pathology of internal organs was observed. The parasites Ichthyophthirius multifiliis and Ichthyobodo necator were observed in skin samples of a proportion of naïve challenged fish and in Trojans but not in control fish. The presence of these and other known fish pathogens in the skin of PSD-fish was confirmed by high-throughput sequencing analysis. In summary, we have demonstrated that PSD is a transmissible condition. However, even though a number of known fish pathogens were identified in the skin tissues of PSD-fish, the actual causative infectious agent(s) remain(s) unknown.

Gaffkaemia, caused by Aerococcus viridans var. homari, causes fatal infections in Homarus spp. (clawed lobsters). Despite its high economic significance to the lobster fisheries in the USA and northern Europe, data on its prevalence in captured and wild populations, particularly in Europe, is scarce. Following an outbreak of gaffkaemia in a European lobster holding facility in South Wales (UK), a base-line survey was conducted for gaffkaemia in wild populations of European lobster Homarus gammarus around the coast of England and Wales. In addition, isolates recovered from the original outbreak and the survey were typed using pulsed-field gel electrophoresis (PFGE) and compared with previously characterised isolates from the USA, UK and Canada. Locally caught H. gammarus were sampled at 30 sites from around the coast of England and Wales between March 2006 and October 2008. Results confirmed that the prevalence of gaffkaemia in populations of H. gammarus was low, with only 9 positive isolates recovered from 952 samples examined. PFGE analysis showed that the isolates from the outbreak investigation shared the same pulsotype as A. viridans var. homari isolates from the USA, Norway and Canada, as well as an isolate (NCIMB 1119) reportedly recovered from an outbreak of European lobsters in England in the 1960s. This confirms earlier studies that suggest virulent strains of A. viridans var. homari show very limited geographical or temporal genetic variation and were introduced into the UK with American lobsters H. americanus.

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.

Background Infectious Pancreatic Necrosis (IPN) is a highly contagious birnavirus disease of farmed salmonid fish, which often causes high levels of morbidity and mortality. A large host genetic component to resistance has been previously described for Atlantic salmon (Salmo salar L.), which mediates high mortality rates in some families and zero mortality in others. However, the molecular and immunological basis for this resistance is not yet fully known. This manuscript describes a global comparison of the gene expression profiles of resistant and susceptible Atlantic salmon fry following challenge with the IPN virus. Results Salmon fry from two IPNV-resistant and two IPNV-susceptible full sibling families were challenged with the virus and sampled at 1 day, 7 days and 20 days post-challenge. Significant viral titre was observed in both resistant and susceptible fish at all timepoints, although generally at higher levels in susceptible fish. Gene expression profiles combined with gene ontology and pathway analyses demonstrated that while a clear immune response was observed in both resistant and susceptible fish, there were striking differences between the two phenotypes. The susceptible fish showed marked up-regulation of genes related to cytokine activity and inflammatory response that evidently failed to protect against the virus. In contrast, the resistant fish demonstrated a less pronounced immune response including up-regulation of genes relating to the M2 macrophage system. Conclusions While only the susceptible phenotype shows appreciable mortality levels, both resistant and susceptible fish can become infected with IPNV. Susceptible fish are characterized by a much larger, yet ineffective, immune response, largely related to cytokine and inflammatory systems. Resistant fish demonstrate a more moderate, putative macrophage-mediated inflammatory response, which may contribute to their survival. Electronic supplementary material The online version of this article (doi:10.1186/s12864-016-2600-y) contains supplementary material, which is available to authorized users.

Disposal of fish by-products in the European Community must comply with Regulation (EC) No 1069/2009 which categorizes animal by-products according to risk, and specifies methods of disposal of by-products according to that risk. There is provision under the regulation for composting or ensiling to be used for by-products from aquatic animals. Biosecurity considerations require knowledge of the parameters of time and temperature, or time and pH, required to inactivate any fish pathogens that may be present. To provide those data, we undertook laboratory studies on the inactivation of a number of fish pathogenic viruses and bacteria at 60 °C, pH 4.0 and pH 12.0 as a preliminary to conducting subsequent trials with the most resistant viruses and bacteria in fish tissues. The most resistant bacterium to 60 °C, pH 4.0 as well as pH 12.0 was Lactococcus garvieae. Its concentration was reduced to the level of sensitivity of the test after 24-48 h exposure to 60 °C, but it survived for at least 7 days at pH 4.0 and 14 days at pH 12.0. The most resistant virus to 60 °C was infectious pancreatic necrosis virus, and to pH 12.0 was infectious salmon anaemia virus. The majority of the viruses tested survived exposure to pH 4.0 for up to 28 days. The results suggest that the process of acid ensiling alone is not an effective method for the inactivation of many viral and bacterial pathogens, and fish by-products would need further treatment by a method approved under the regulation following ensiling, whereas alkaline or heat treatment are likely to provide an increased degree of biosecurity for on-farm processing of mortalities.

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.

Biotype 2 (BT2) variants of the bacterium Yersinia ruckeri are an increasing disease problem in U.S. and European aquaculture and have been characterized as serovar 1 isolates that lack both peritrichous flagella and secreted phospholipase activity. The emergence of this biotype has been associated with an increased frequency of enteric redmouth disease (ERM) outbreaks in previously vaccinated salmonid fish. In this study, four independent specific natural mutations that cause the loss of both motility and secreted lipase activity were identified in BT2 strains from the United States, United Kingdom, and mainland Europe. Each of these was a unique mutation in either fliR , flhA , or flhB , all of which are genes predicted to encode essential components of the flagellar secretion apparatus. Our results demonstrate the existence of independent mutations leading to the BT2 phenotype; thus, this phenotype has emerged separately at least four times. In addition, BT2 strains from the United Kingdom were shown to have the same mutant allele found in U.S. BT2 strains, suggesting a common origin of this BT2 lineage. This differentiation of distinct BT2 lineages is of critical importance for the development and validation of alternative vaccines or other treatment strategies intended for the control of BT2 strains.

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.

BACKGROUND: Antimicrobial resistance remains a growing and significant concern in human and veterinary medicine. Current laboratory methods for the detection and surveillance of antimicrobial resistant bacteria are limited in their effectiveness and scope. With the rapidly developing field of whole genome sequencing beginning to be utilised in clinical practice, the ability to interrogate sequencing data quickly and easily for the presence of antimicrobial resistance genes will become increasingly important and useful for informing clinical decisions. Additionally, use of such tools will provide insight into the dynamics of antimicrobial resistance genes in metagenomic samples such as those used in environmental monitoring. RESULTS: Here we present the Search Engine for Antimicrobial Resistance (SEAR), a pipeline and web interface for detection of horizontally acquired antimicrobial resistance genes in raw sequencing data. The pipeline provides gene information, abundance estimation and the reconstructed sequence of antimicrobial resistance genes; it also provides web links to additional information on each gene. The pipeline utilises clustering and read mapping to annotate full-length genes relative to a user-defined database. It also uses local alignment of annotated genes to a range of online databases to provide additional information. We demonstrate SEAR's application in the detection and abundance estimation of antimicrobial resistance genes in two novel environmental metagenomes, 32 human faecal microbiome datasets and 126 clinical isolates of Shigella sonnei. CONCLUSIONS: We have developed a pipeline that contributes to the improved capacity for antimicrobial resistance detection afforded by next generation sequencing technologies, allowing for rapid detection of antimicrobial resistance genes directly from sequencing data. SEAR uses raw sequencing data via an intuitive interface so can be run rapidly without requiring advanced bioinformatic skills or resources. Finally, we show that SEAR is effective in detecting antimicrobial resistance genes in metagenomic and isolate sequencing data from both environmental metagenomes and sequencing data from clinical isolates.

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.

Epidemiological cut-off values were developed for application to antibiotic susceptibility data for Flavobacterium psychrophilum generated by standard CLSI test protocols. The MIC values for ten antibiotic agents against Flavobacterium psychrophilum were determined in two laboratories. For five antibiotics, the data sets were of sufficient quality and quantity to allow the setting of valid epidemiological cut-off values. For these agents, the cutoff values, calculated by the application of the statistically based normalized resistance interpretation method, were

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.

To the Editor: Doctor fish (Garra rufa) are freshwater cyprinid fish that naturally inhabit river basins in central Eurasia. They are widely used in the health and beauty industries in foot spas for ichthyotherapy (Kangal fish therapy or doctor fish therapy) (Figure; Technical Appendix Figure 1) (1). During these sessions, patients immerse their feet or their entire bodies in the spas, allowing the fish to feed on dead skin for cosmetic reasons or for control of psoriasis, eczema, and other skin conditions. Figure Doctor fish surrounding foot during ichthyotherapy. A survey during the spring of 2011 identified 279 fish spas in the United Kingdom, and the number has probably increased since then (1). The Fish Health Inspectorate of the Centre for Environment, Fisheries & Aquaculture Science estimates that each week 15,000–20,000 G. rufa fish are imported from Indonesia and other countries in Asia into the United Kingdom through London Heathrow Airport (the main border inspection post for the import of live fish). However, ichthyotherapy has now reportedly been banned in several US states and Canada provinces because of sanitary concerns (1). In the United Kingdom, a limited number of infections after fish pedicures have been reported (1). Unfortunately, little is known about the types of bacteria and other potential pathogens that might be carried by these fish and the potential risks that they might pose to customers or to ornamental and native fish. On April 12, 2011, the Fish Heath Inspectorate investigated a report of a disease outbreak among 6,000 G. rufa fish from Indonesia that had been supplied to UK pedicure spas. Affected fish showed clinical signs of exophthalmia and of hemorrhage around the gills, mouth, and abdomen. More than 95% of the fish died before the remaining fish were euthanized. Histopathologic examinations identified systemic bacterial infections with small gram-positive cocci, mostly in the kidneys, spleen, and liver. Bacterial isolates cultured from affected fish were identified as Streptococcus agalactiae (group B Streptococcus) according to a combination of biochemical test results (API Strep; bioMerieux, Marcy l’Etoile, France), Lancefield grouping with serotype B (Oxoid Limited, Basingstoke, UK), and molecular (partial 16S rRNA gene sequencing) testing methods. Multilocus sequence typing of a representative isolate (11013; Technical Appendix Table) (2) indicated that it was a sequence type (ST) 261 S. agalactiae strain (http://pubmlst.org/sagalactiae). This same ST261 profile was first identified in an isolate (ATCC 51487) from a diseased tilapia in Israel (3). The clinical appearance of the disease and the diagnostic results suggested that S. agalactiae was the causative agent of the fish illness and deaths. To determine whether S. agalactiae and other bacterial pathogens might be carried more widely by these fish, from May 5, 2011, through June 30, 2011, the Fish Health Inspectorate of the Centre for Environment, Fisheries & Aquaculture Science visited Heathrow Airport 5 times to intercept and sample consignments of G. rufa from Indonesia. A taxonomically diverse range of bacteria were identified (Technical Appendix Table, Figure 2), including a variety of human pathogens capable of causing invasive soft tissue infections. These pathogens included Aeromonas spp (4), potentially pathogenic clinical-type Vibrio vulnificus isolates (Technical Appendix Figure 2) (5), non–serotype O1 or O139 cholera toxin–negative V. cholerae isolates (Technical Appendix Figure 2) (6), Mycobacteria (7), and S. agalactiae (3,8). Isolates were resistant to a variety of antimicrobial drugs, including tetracyclines, fluoroquinolones, and aminoglycosides (Technical Appendix Table). Other studies have also reported high levels of multidrug resistance in bacteria associated with imported ornamental fish (9). Water is a well-recognized source of bacterial skin infections in humans. V. vulnificus can cause wound infections and primary septicemia, resulting in high mortality rates, especially among persons with predisposing risk conditions (e.g., liver disease, diabetes, or impaired immune function) (5). S. agalactiae is a common cause of skin and soft tissue infections, especially in older adults and those with chronic diseases such as diabetes mellitus (8). Although S. agalactiae ST261 is not considered to be one of the genotypes typically associated with invasive disease in humans (3), a fish-adapted strain could eventually take advantage of the opportunity afforded by repeated exposure and thereby also affect humans. Additionally, Mycobacteria spp. can occasionally cause disease in humans through contact with fish (M. marinum), and pedicure treatments have previously been associated with M. fortuitum infections (10). Recently, the risks associated with exposure to G. rufa fish were reported to be low (1). To date, there are only a limited number of reports of patients who might have been infected by this exposure route (1). However, our study raises some concerns over the extent that these fish, or their transport water, might harbor potential zoonotic disease pathogens of clinical relevance. In particular, patients with underlying conditions (such as diabetes mellitus or immunosuppression) should be discouraged from undertaking such treatments, especially if they have obvious breaks in the skin or abrasions. This risk can probably be reduced by use of certified disease-free fish reared in controlled facilities under high standards of husbandry and welfare.

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.