Understanding of risk factors for parasite introduction and amplification within fish farms is crucial to design effective biosecurity and control management strategies but currently, quantitative data on the relevance of such risk factors is largely absent. We have designed the questionnaires for stakeholders to characterize the risk factors and estimate their relevance for infections causing major economic burden on gilthead seabream ( Sparus aurata ) and European seabass ( Dicentrarchus labrax ) production: the myxozoan Enteromyxum leei , the monogenean Sparicotyle chrysophrii , the crustacean Ceratothoa oestroides and the dinoflagellate Amyloodinium ocellatum . Twenty-two experts (Belgium, Croatia, Greece, Italy, Norway, and Spain) were invited to participate in an online questionnaire, followed by a physical meeting to discuss the most probable values for parameter estimates. Quantitative estimates were obtained for the relevance of risk routes of parasite spread (e.g., the likelihood of parasite transmission from an infected to an uninfected net cage 5 m away was estimated to be 90% for E. leei ), and the relevance of management procedures and environmental factors that may have an impact on the probability of infection to lead to disease (e.g., where mortalities are removed at > 5-day intervals, the likelihood that infection will lead to disease was 30% for S. chrysophrii ). These quantitative estimates were appraised, including the delay in reaching harvest size, or cumulative mortality during phases of production, being essential for calculation of the burden of these diseases in aquaculture. The data presented are highly valuable to the development of economically viable biosecurity and specific integrated pest management strategies.

Viral haemorrhagic septicaemia virus (VHSV) is a fish disease notifiable to the World Organisation for Animal Health. The United Kingdom is currently free from VHSV, and the introduction and onward spread of this disease could cause major economic losses in aquaculture facilities. Legislation in Great Britain requires that imports of live fish for aquaculture purposes into declared disease-free areas are of equivalent disease-free status. However, conditions on fish products are less stringent, whereby eviscerated fish or fillets can be transported from areas with disease to areas declared disease-free. Market-size rainbow trout were experimentally infected with VHSV to investigate two important factors relevant for pathogen introduction and transmission: (1) VHSV shedding, quantified by daily assessment of viral titres in tank water samples, and (2) VHSV concentrations in liquid and solid processing waste. Evisceration and filleting preclinical fish, maceration, and wastewater separation processes within a facility were mimicked, and VHSV was quantified in each fraction of the wastewater. Shedding was detected 25 hr post-challenge. Levels increased daily to peak on day 5 post-challenge, with a calculated average titre of 1.35 × 10 3 TCID 50  mL −1  kg −1 fish, 1 day before clinical signs of disease. Preclinical fish contained virus levels in their kidney, skin, and muscle >10 7 TCID 50  g −1 . The fish had significantly higher levels in the kidney, and evisceration led to higher VHSV concentrations in the waste compared to filleting. However, there was no significant difference in levels in wastewater released from the two processes after the removal of solids, even when macerated; average titres were >10 4 TCID 50  mL −1 . The quantities of VHSV from shedding and processing can be utilised when modelling transmission and undertaking more accurate risk assessments for imports and processing of commodities, with the ultimate aim of reducing the global risk of disease from international trade and processing.

Oomycetes of the genus Saprolegnia are widespread in freshwater environment and are among the main pathogens causing economic losses in salmonid aquaculture. Infections by mycotic agents in fish farming are generally considered to result from chronic stress and poor fish condition associated with water quality problems, adverse environmental conditions, frequent/rough/incorrect handling, concurrent infections, physiological changes associated with reproduction and immunocompromised animals. To identify risk factors for Saprolegnia infections in trout and Atlantic salmon farming, longitudinal studies were carried out in different Italian, Spanish, and Scottish fish farms. Prevalence of saprolegniosis and fish mortality were monitored over time and statistically analysed with respect to husbandry and environmental factors. Overall, statistical results by production cycle (trout vs salmon farming) and by country indicate that the prevalence of Saprolegnia may be influenced by peculiarities of the culture system and farming environment. Nevertheless, a specific set of parameters, including lower water temperature, and handling procedures increased Saprolegnia prevalence in all the considered farms. Particularly, in trout farms Saprolegnia infections represented an important contribution to mortality, and prevalence was influenced by water temperature and pH, and by fish density within the tanks. Similarly, temperature and water quality were the main factors influencing the prevalence of Saprolegnia in Atlantic salmon farms. Moreover, molecular analyses confirmed the role of S. parasitica as the main pathogenic oomycete in trout and salmon farming in the considered countries. The identification of risk factors for introduction and increase of Saprolegnia infection in fish farms will allow the correct design of biosecurity and pathogen control strategies.

Viral haemorrhagic septicaemia virus (VHSV) and infectious haematopoietic necrosis virus (IHNV) are important viral pathogens posing a serious threat to salmonid fish. Survival of two isolates of IHNV and one of VHSV was assessed at temperatures ranging from 4 to 25°C: (a) after drying on stainless steel, (b) in cell culture medium, (c) in filtered river water, (d) in unfiltered river water, and (e) survival, adsorption and desorption in river sediment and five typical soil types. The viruses survived 1 hr to > 84 days depending on the conditions. Survival was inversely related to temperature and organic and inorganic content. Both viruses remained infectious after being dried on stainless steel for several weeks highlighting the risk of mechanical transmission and persistence in a dry environment. Both adsorbed to the soils from the river water inoculum, with titres between 5.56x104 and 2.58x108 TCID50 /ml after 1 hr. Clay soils adsorbed the least virus but had the greatest decrease in the river water inoculum (undetectable in ≤ 1 hr), and there was no desorption. Virus desorbed from the other soils into the surrounding water at different rates dependant on soil type (longest desorption was from chalk loam and sandy soil-detected at 28 days). When desorption was no longer detectable, virus persisted, adsorbed to the soil and remained infectious (the longest adsorption was detected in clay loam for ≥ 49 days, but all the viruses adsorbed to soils were likely to have survived longer than that detected, based on their rate of decay). The long survival of the viruses, particularly at cooler temperatures, highlights the risk of survival in the environment and waterborne spread. The data presented here are highly relevant for assessing risk of pathogen introduction via fomites (stainless steel) and for deciding on best control measures in the context of disease outbreaks.

Aphanomyces astaci causes crayfish plague, which is a devastating disease of European freshwater crayfish. The likely first introduction of A. astaci into Europe was in the mid-19th century in Italy, presumably with the introduction of North American crayfish. These crayfish can carry A. astaci in their cuticle as a benign infection. Aphanomyces astaci rapidly spread across Europe causing the decline of the highly susceptible indigenous crayfish species. Random amplified polymorphic DNA-PCR analysis of A. astaci pure cultures characterized five genotype groups (A, B, C, D and E). Current A. astaci genotyping techniques (microsatellites and genotype-specific regions, both targeting nuclear DNA) can be applied directly to DNA extracted from infected cuticles but require high infection levels. Therefore, they are not suitable for genotyping benign infections in North American crayfish (carriers). In the present study, we combine bioinformatics and molecular biology techniques to develop A. astaci genotyping molecular markers that target the mitochondrial DNA, increasing the sensitivity of the genotyping tools. The assays were validated on DNA extracts of A. astaci pure cultures, crayfish tissue extractions from crayfish plague outbreaks and tissue extractions from North American carriers. We demonstrate the presence of A. astaci genotype groups A and B in UK waters.

The functional mechanism of anti-merozoite antibodies.Antibodies to merozoite surface proteins can mediate several eff ector mechanisms, including complement fi xation due to cytophillic antibodies that result in merozoite lysis of C3b opsonization; inhibition of merozoite invasion into the RBC; phagocytosis of IgG-opsonized merozoites; production of reactive oxygen species (ROS) or Nitric oxide (NO) in response to opsonized parasites

The oomycete Aphanomyces astaci causes crayfish plague, the most important disease of European freshwater crayfish species. Presumably introduced into Europe 150 years ago with the import of North American crayfish, A. astaci is highly pathogenic to European crayfish species. Five genotypes (A, B, C, D, and E) have been defined based on random amplified polymorphic DNA analysis (RAPD-PCR) from A. astaci pure cultures. The distinction of genotypes is an essential tool to conduct molecular epidemiological studies on crayfish plague and it has been used to clarify and better understand the history and spread of this disease in Europe. Whereas RAPD-PCR requires DNA from pure culture isolates, the development of genotyping tools that can be applied to DNA extracted from clinical samples allows a much wider application of genotyping studies, including revisiting historic samples. In this study, we present a new approach that adds to currently available methods for genotyping A. astaci strains directly from clinical crayfish samples. Whole-genome sequencing of A. astaci strains representing all currently known genotypes was employed, genomic regions unique to the respective genotype identified, and a PCR-based genotyping assay designed, which focuses on the presence/absence of PCR product after amplification with the genotype-specific primers. Our diagnostic methodology was tested using DNA extracts from pure A. astaci cultures, other Aphanomyces species and additional oomycetes, samples from a recent Italian crayfish plague outbreak and additional historical samples available in the Centre for Environment, Fisheries and Aquaculture Science laboratory. The new markers were reliable for pure culture and clinical samples from a recent outbreak and successfully discriminated genotype A, B, D, and E. The marker for genotype C required an additional sequencing step of the generated PCR product to confirm genotype.

The European Union Council Directive 2006/88/EC requires that risk-based surveillance (RBS) for listed aquatic animal diseases is applied to all aquaculture production businesses. The principle behind this is the efficient use of resources directed towards high-risk farm categories, animal types and geographic areas. To achieve this requirement, fish and shellfish farms must be ranked according to their risk of disease introduction and spread. We present a method to risk rank shellfish farming areas based on the risk of disease introduction and spread and demonstrate how the approach was applied in 45 shellfish farming areas in England and Wales. Ten parameters were used to inform the risk model, which were grouped into four risk themes based on related pathways for transmission of pathogens: (i) live animal movement, (ii) transmission via water, (iii) short distance mechanical spread (birds) and (iv) long distance mechanical spread (vessels). Weights (informed by expert knowledge) were applied both to individual parameters and to risk themes for introduction and spread to reflect their relative importance. A spreadsheet model was developed to determine quantitative scores for the risk of pathogen introduction and risk of pathogen spread for each shellfish farming area. These scores were used to independently rank areas for risk of introduction and for risk of spread. Thresholds were set to establish risk categories (low, medium and high) for introduction and spread based on risk scores. Risk categories for introduction and spread for each area were combined to provide overall risk categories to inform a risk-based surveillance programme directed at the area level. Applying the combined risk category designation framework for risk of introduction and spread suggested by European Commission guidance for risk-based surveillance, 4, 10 and 31 areas were classified as high, medium and low risk, respectively.

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The identification of risk factors for parasite introduction into and amplification within fish farms is crucial in order to design effective biosecurity and parasite control management strategies. Currently, there is a substantial lack of knowledge about such risk factors. A structured review of the available literature on risk factors for selected parasites was undertaken with the aim to bring together relevant knowledge on characteristics of host and parasite biology and risk factors for infection and amplification of parasites on fish farms. This process helped to assess the level of data available and identification of data gaps. Questionnaires were designed to provide parameter estimates on the relevance of risk factors for initial parasite infection and risk factors for infection causing impact for infection of gilthead seabream (Sparus aurata) with Enteromyxum leei and Sparicotyle chrysophrii, and of European seabass (Dicentrarchus labrax) with Ceratothoa oestroides and Amyloodinium ocellatum. Separate questionnaires were developed for each of parasite. Highly experienced experts were then invited to participate in a two-stage consultation process: participation in an online questionnaire, followed by a physical meeting. The responses obtained provide quantitative estimates for the relevance of risk routes of infection with the respective parasites, the relevance of management procedures for reducing or increasing mortality due to parasite infection, relevance of environmental factors, such as water exchange rate. Examples of risk factors that were relevant for development of infection causing impact were frequency of removal of mortalities from rearing units, number of cage farm sites in a production area. Management procedures that were identified to significantly reduce losses are separation of year classes during production, and fallowing. Furthermore, important quantitative estimates of impacts of parasite infection on aquaculture production in terms of delay in reaching harvest size or cumulative mortality during certain phases of production were obtained. The literature reviews and outcomes from the expert consultations are now forming the basis for planning further data collection through field studies. The information brought together will also feed into the development of biosecurity and integrated pest management strategies and assessments of the most economic management strategies of parasite infections. Funding of presentation: This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No 634429. This output reflects the views only of the author(s), and the European Union cannot be held responsible for any use which may be made of the information contained therein.

The crayfish plague pathogen (Aphanomyces astaci) causes mass mortalities of European crayfish when transmitted from its original North American crayfish hosts. Little is known, however, about interspecific transmission of the pathogen between different American crayfish species, although evidence from trade of ornamental crayfish suggests this may happen in captivity. We screened signal and virile crayfish for A. astaci at allopatric and sympatric sites in a UK river. Whilst the pathogen was detected in signal crayfish from both sites, infected virile crayfish were only found in sympatry. Genotyping of A. astaci from virile crayfish suggested the presence of a strain related to one infecting British signal crayfish. We conclude that virile crayfish likely contracted A. astaci interspecifically from infected signal crayfish. Interspecific transmission of A. astaci strains differing in virulence between American carrier species may influence the spread of this pathogen in open waters with potential exacerbated effects on native European crayfish.

SUMMARY The crayfish plague agent, Aphanomyces astaci , has spread throughout Europe, causing a significant decline in native European crayfish. The introduction and dissemination of this pathogen is attributed to the spread of invasive North American crayfish, which can act as carriers for A. astaci . As native European crayfish often succumb to infection with A. astaci , determining the prevalence of this pathogen in non-native crayfish is vital to prioritize native crayfish populations for managed translocation. In the current study, 23 populations of invasive signal crayfish ( Pacifastacus leniusculus ) from the UK were tested for A. astaci presence using quantitative PCR. Altogether, 13 out of 23 (56·5%) populations were found to be infected, and pathogen prevalence within infected sites varied from 3 to 80%. Microsatellite pathogen genotyping revealed that at least one UK signal crayfish population was infected with the A. astaci genotype group B, known to include virulent strains. Based on recent crayfish distribution records and the average rate of signal crayfish population dispersal, we identified one native white-clawed crayfish ( Austropotamobius pallipes ) population predicted to come into contact with infected signal crayfish within 5 years. This population should be considered as a priority for translocation.

Aquatic animal diseases are a major constraint for increasing aquaculture production. Understanding the contribution of pathogen spread from infected aquaculture sites is critical in devising control measures in the event of an outbreak. We have reviewed the available literature on the persistence in the aquatic environment of several important viral pathogens of fish and crustaceans. These include infectious haematopoietic necrosis virus, viral haemorrhagic septicaemia virus, infectious salmon anaemia virus, koi herpes virus, epizootic haematopoietic necrosis virus and infectious pancreatic necrosis virus, white spot syndrome virus (WSSV), Taura syndrome virus and yellow head virus. Some trends were common to all viruses: (i) viability declined with increasing temperature (at temperatures above 0 degrees C); (ii) higher biological loading in water correlated with reduction in detectable viable viruses; and (iii) virus decay in water is a function of time. Most aquatic animal viruses (AAVs) remained viable for several days or weeks. WSSV is particularly stable. Comparison of studies investigating survival parameters was sometimes difficult because of the different methods employed and different ways in which the data were presented. Data gaps are identified and experimental methods employed for testing critically assessed. The information presented in this review is directly relevant to design effective control measures for AAVs and to explore measures that reduce the economic impact of disease caused by these important pathogens.

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.

Infectious hematopoietic necrosis virus (IHNV, Rhabdoviridae), is the causative agent of infectious hematopoietic necrosis (IHN), a disease notifiable to the World Organisation for Animal Health, and various countries and trading areas (including the European Union). IHNV is an economically important pathogen causing clinical disease and mortalities in a wide variety of salmonid species, including the main salmonid species produced in aquaculture, Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss). We reviewed the scientific literature on IHNV on a range of topics, including geographic distribution; host range; conditions required for infection and clinical disease; minimum infectious dose; subclinical infection; shedding of virus by infected fish; transmission via eggs; diagnostic tests; pathogen load and survival of IHNV in host tissues. This information is required for a range of purposes including import risk assessments; parameterisation of disease models; for surveillance planning; and evaluation of the chances of eradication of the pathogen to name just a few. The review focuses on issues that are of relevance for the European context, but many of the data summarised have relevance to IHN globally. Examples for application of the information is presented and data gaps highlighted.

A qualitative import risk assessment was undertaken to assess the likelihood of introduction and establishment of viral haemorrhagic septicaemia virus (VHSV) genotype 1a in England and Wales (E&W), via the processing of imported rainbow trout (Oncorhynchus mykiss) carcasses from continental Europe. The likelihood was estimated for one import from an infected farm. Four main routes by which susceptible populations could be exposed to VHSV via processing waste were considered: (i) run-off from solid waste to watercourses, (ii) contamination of birds or rodents with VHSV by scavenging solid waste, (iii) discharge of liquid waste to mains drainage, and (iv) discharge of liquid waste directly to watercourses. Data on the biophysical characteristics of VHSV, its epidemiology, fish processing practices and waste management were collected. Likelihoods for each step of the four pathways were estimated. Pathway 4 (discharge of liquid waste to a watercourse) was judged as the most likely to result in infection of susceptible individuals. Levels of virus entering the aquatic environment via pathways 1-3 were judged to be many times lower than pathway 4 due mainly to the treatment of solid waste (pathways 1 and 2) and high levels of dilution (pathways 1, 2 and 3). Thirty-four trout farms process fish, of which seven have imported carcasses for processing. Compared with other processing facilities, on-farm processing results in a higher likelihood of VHSV exposure and establishment via all four pathways. Data availability was an issue; the analysis was particularly constrained by a lack of data on the prevalence of VHSV in Europe, volume of trade of carcasses into the UK and processing practices in E&W. It was concluded that the threat of VHSV introduction into E&W could be reduced by treatment of liquid effluent from processing plants and by sourcing carcasses for on-farm processing only from approved VHSV free areas.

North American crayfish species as hosts for the crayfish plague pathogen Aphanomyces astaci contribute to the decline of native European crayfish populations. At least six American crayfish species have been reported in the Netherlands but the presence of this pathogenic oomycete with substantial conservational impact has not yet been confirmed in the country. We evaluated A. astaci prevalence in Dutch populations of six alien crustaceans using species-specific quantitative PCR. These included three confirmed crayfish carriers (Orconectes limosus, Pacifastacus leniusculus, Procambarus clarkii), two recently introduced but yet unstudied crayfish (Orconectes cf. virilis, Procambarus cf. acutus), and a catadromous crab Eriocheir sinensis. Moderate levels of infection were observed in some populations of O. limosus and P. leniusculus. Positive results were also obtained for E. sinensis and two Dutch populations of O. cf. virilis. English population of the latter species was also found infected, confirming this taxon as another A. astaci carrier in European waters. In contrast, Dutch P. clarkii seem only sporadically infected, and the pathogen was not yet detected in P. cf. acutus. Our study is the first confirmation of crayfish plague infections in the Netherlands and demonstrates substantial variation in A. astaci prevalence among potential hosts within a single region, a pattern possibly linked to their introduction history and coexistence.