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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.

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.

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.

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.

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.

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.

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.

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.

Background Microbial diseases cause considerable economic losses in aquaculture and new infection control measures often rely on a better understanding of pathogenicity. However, disease studies performed in fish hosts often require specialist infrastructure (e.g., aquaria), adherence to strict legislation and do not permit high-throughput approaches; these reasons justify the development of alternative hosts. This study aimed to validate the use of larvae of the greater wax moth (Galleria mellonella) to investigate virulence of the important fish pathogen, Vibrio anguillarum. Results Using 11 wild-type isolates of V. anguillarum, these bacteria killed larvae in a dose-dependent manner and replicated inside the haemolymph, but infected larvae were rescued by antibiotic therapy. Crucially, virulence correlated significantly and positively in larva and Atlantic salmon (Salmo salar) infection models. Challenge studies with mutants knocked out for single virulence determinants confirmed conserved roles in larva and fish infections in some cases (pJM1 plasmid, rtxA), but not all (empA, flaA, flaE). Conclusions The G. mellonella model is simple, more ethically acceptable than experiments on vertebrates and, crucially, does not necessitate liquid systems, which reduces infrastructure requirements and biohazard risks associated with contaminated water. The G. mellonella model may aid our understanding of microbial pathogens in aquaculture and lead to the timely introduction of new effective remedies for infectious diseases, while adhering to the principles of replacement, reduction and refinement (3Rs) and considerably reducing the number of vertebrates used in such studies. Electronic supplementary material The online version of this article (doi:10.1186/s12866-015-0466-9) contains supplementary material, which is available to authorized users.

Background Bacterial kidney disease (BKD), caused by Renibacterium salmoninarum, is a bacterial disease of fish, which is both geographically widespread and difficult to control. Previously, application of various molecular typing methods has failed to reliably discriminate between R. salmoninarum isolates originating from different host species and geographic areas. The current study aimed to utilize multilocus variable number tandem repeats (VNTR) to investigate inter-strain variation of R. salmoninarum to establish whether host-specific populations exist in Atlantic salmon and rainbow trout respectively. Such information would be valuable in risk assessment of transmission of R. salmoninarum in a multispecies aquaculture environment. Results The present analysis utilizing sixteen VNTRs distinguished 17 different haplotypes amongst 41 R. salmoninarum isolates originating from Atlantic salmon and rainbow trout in Scotland, Norway and the US. The VNTR typing system revealed two well supported groups of R. salmoninarum haplotypes. The first group included R. salmoninarum isolates originating from both Atlantic salmon and rainbow trout circulating in Scottish and Norwegian aquaculture, in addition to the type strain ATCC33209T originating from Chinook salmon in North America. The second group comprised isolates found exclusively in Atlantic salmon, of mainly wild origin, including isolates NCIB1114 and NCIB1116 associated with the original Dee disease in Scotland. Conclusions The present study confirmed that VNTR analysis can be successfully applied to discriminate R. salmoninarum strains. There was no clear distinction between isolates originating from Atlantic salmon and rainbow trout as several haplotypes in group 1 clustered together R. salmoninarum isolates from both species. These findings indicate a potential exchange of pathogens between Atlantic salmon and rainbow trout in Scottish and Norwegian aquaculture during the last 20 years. In a scenario of expansion of rainbow trout farming into the marine environment, appropriate biosecurity measures to minimize disease occurrence are advised. The present results also suggest that R. salmoninarum isolates circulating in European aquaculture over the last 20 years are genetically distant to the wild strains originally causing BKD in the rivers Dee and Spey.