An assessment of health management and biosecurity procedures in marine fish farming in Spain

Marine fish farming in Spain has experienced problems of performance due to losses caused by infectious diseases. Biosecurity and health management are identified by the Food and Agriculture Organization (FAO) as current priorities for proper aquaculture governance. However, they both transcend the responsibility of farmers and require significant resources, concerted action and cooperation. This study presents the analysis of biosecurity practices on marine fish farms, through a questionnaire-based survey on biosecurity procedures and an analysis of health management practices for different stakeholders. The Strengths, Weaknesses, Opportunities, and Threats (SWOT) technique was implemented, which identified the important threats and weaknesses faced by the sector, such as the risk of direct disease transmission between farms, the high likelihood of importing diseases through juvenile shipments, the chronic lack of communication between stakeholders, and the deficient coordination of health strategies. Strengths included awareness of prevention measures and the availability of expertize of health experts at most levels. On the other hand, the availability of experts together with the need to adapt governance to the current production systems were seen as opportunities. Health management measures themselves were actually already found to be adapted to the type of production but they varied between companies (i.e. categorization and diagnosis of mortalities). Nevertheless, the quality of expertize along the value chain provided by private and public laboratories, research institutes, Health Protection Groups, companies and veterinarians was noteworthy. However, there was still a need for all stakeholders involved in marine fish health to improve diagnostics, provide epidemiological information, biosecurity and prevention measures, as well as to promote transparency for better health governance.info:eu-repo/semantics/publishedVersio

Detection of isothermally amplified ostreid herpesvirus 1 DNA in Pacific oyster (Crassostrea gigas) using a miniaturised electrochemical biosensor

Given the threat that ostreid herpesvirus 1 (OsHV-1) poses to shellfish aquaculture, the need for rapid, user-friendly and cost-effective methods to detect this marine pathogen and minimise its impact is evident. In this work, an electrochemical biosensor for the detection of OsHV-1 based on isothermal recombinase polymerase amplification (RPA) was developed. The system was first tested and optimised on maleimide microtitre plates as a proof-of-concept, before being implemented on miniaturised gold electrodes. Amperometric detection of the isothermally amplified product was achieved through a sandwich hybridisation assay with an immobilised thiolated capture probe and a horseradish peroxidase (HRP)-labelled reporter probe. Calibration curves were constructed using PCR-amplified OsHV-1 DNA, achieving a limit of detection of 207 OsHV-1 target copies. The biosensor was applied to the analysis of 16 oyster samples from an infectivity experiment, and results were compared with those obtained by qPCR analysis, showing a strong degree of correlation (r = 0.988). The simplicity, rapidity, cost-effectiveness and potential for in-situ testing with the developed biosensor provide a valuable tool for the detection of OsHV-1 in aquaculture facilities, improving their management.info:eu-repo/semantics/acceptedVersio

Vibrio mediterranei, a Potential Emerging Pathogen of Marine Fauna: Investigation of pathogenicity using a bacterial challenge in Pinna nobilis and development of a species‐specific PCR

Aims Extreme mortality events affecting Pinna nobilis, some associated to Vibrio mediterranei, have depleted many populations of this bivalve. The objective of this study was to demonstrate pathogenicity of V. mediterranei in the host P. nobilis by performing a bacterial challenge in P. nobilis to understand if V. mediterranei has specific virulence in this host. To assist this objective, a secondary objective was to develop a species‐specific DNA diagnostic test. Methods and Results P. nobilis collected from local bays were used in a challenge experiment with V. mediterranei (strain IRTA18‐108). Virulence in the host background of P. nobilis was demonstrated at doses of 103 CFUs / animal. An alignment of published Vibrio spp. atpA sequences was used to design V. mediterranei ‐specific primers. Further, data mining of published literature and V. mediterranei genomes identified multiple virulence‐related genes (vir genes) from which specific primers were designed for PCR detection of selected genes. Conclusion V. mediterranei strain IRTA18‐108 is pathogenic in the host P. nobilis . The virulence genes sod, rtx , and mshA were identified in this strain. Temperatures of 24ºC or higher appear to trigger onset of virulence. Sensitivity and specificity of the Vm atpA PCR is useful for diagnosis of Vibriosis in shellfish. Significance and Impact of the Study The presence of previously described virulence genes have been confirmed in this strain. The specific Vm atpA PCR assay will aid management of future epizootics of this emerging pathogen of aquatic fauna, and improve surveillance capabilities for mortality events where Vibrios are suspect.info:eu-repo/semantics/acceptedVersio

Do the Escherichia coli European Union shellfish safety standards predict the presence of Arcobacter spp., a potential zoonotic pathogen?

The genus Arcobacter comprises Campylobacter-related species, considered zoonotic emergent pathogens, the presence of which in water has been associated with fecal pollution. Discharges of fecal polluted water into the sea have been considered as one of the main reasons for the presence of Arcobacter in shellfish, and this may represent a risk for public health. In this study, the European Union shellfish food safety criteria based on levels of Escherichia coli were studied in relation to their capacity to predict the presence of Arcobacter species. In addition, the accumulation factor (AF) that measures the concentration ratio between the microbes present in the shellfish and in the water, was also studied for both bacteria. The results show that the presence of E. coli correlated with the presence of the potentially pathogenic species A. butzleri and A. cryaerophilus. However, in 26.1% of the shellfish samples (corresponding to those taken during summer months) E. coli failed to predict the presence of, for instance A. butzleri and A. skirrowii, among other species. In the rest of the samples a significant correlation between the concentration of E. coli and Arcobacter spp. (mussels and oyster; R2 = 0.744) was found. This study indicates that the presence of E. coli can predict the presence of pathogenic Arcobacter species in shellfish samples harvested from water with temperatures lower than 26.2 °C. Consumption of shellfish collected at higher temperatures which may not be permissive to the growth of E. coli but does allow growth of Arcobacter spp., may represent a risk for consumers.info:eu-repo/semantics/acceptedVersio

A mortality event of the venerid bivalve Callista chione (Linnaeus, 1758) in a hatchery system. A case study

Abnormal mortality of the smooth venus clam (Callista chione) was encountered when conditioning these clams in a hatchery system. A histopathological analysis was performed to establish the causes of this mortality episode. Our results showed an increase in rickettsia-like bacteria infection intensity between the individuals collected at the start of the conditioning in the hatchery and those collected during the mortality episode. Husbandry stress most likely increased disease susceptibility and progression in these clams. Rickettsia-like colonies were observed in large numbers in the gills of all individuals examined. Nematopsis sp. spores and rod-shaped basophilic bacteria could also been seen in some of the individuals examined. Microbiological analysis of clam tissue did not reveal the presence of any potentially pathogenic bacteria and all the clams were shown to be free of Perkinsus sp. parasites. The conditioning protocol was adapted from those used for other venerid clams due to the lack of data on this species. These findings highlight the need to perform further studies to evaluate the optimal parameters for C. chione broodstock conditioning

Assessing the economic impact of diseases in Mediterranean grow-out farms culturing European sea bass

The aim of this work is to propose a novel and formal approach to evaluate the direct costs of diseases caused by different pathogens as well as their economic impact on typical Mediterranean grow-out farms culturing European sea bass under different scenarios of production related to the biomass produced (farm size) and the size of the fish produced (production strategy). We employ a deterministic static model to simulate the annual income statement of those facilities to evaluate the direct costs caused by different diseases as well as, through a partial budget and sensitivity analyses, the economic impact of them. An important conclusion of this work is that the profitability and economic viability of sea bass grow-out farms suffering recurrent outbreaks of diseases caused by different pathogens depend on the farm typology (farm size) as well as the decisions taken by owners/investors about the size of the fish produced and sold in the market. Our estimations show that as the larger is the farm and the size of the fish produced, the larger are the direct cost of a disease outbreak. However, the economic impact on the net operating profit is significantly worse as smaller is the farm and smaller the fish produced. The conclusions of this research stress the need for small producers to pay attention and devote resources to prevent and treat disease outbreaks.info:eu-repo/semantics/publishedVersio

Mapping the knowledge of the main diseases affecting sea bass and sea bream in Mediterranean

Good knowledge on the disease situation and its impact on production is a base mechanism for designing health surveillance, risk analysis and biosecurity systems. Mediterranean marine fish farming, as any aquaculture production, is affected by various infectious diseases. However, seabass and seabream, the main produced species, are not listed as susceptible host species for the notifiable pathogens listed in the current EU legislation, which generates a lack of systematic reporting. The results presented in this study come from a survey directly to fish farms (50 hatchery and ongrowing units from 10 Mediterranean countries), with data from 2015‐2017, conducted by the H2020 project MedAID. Seabass showed a higher survival rate (85%) through a production cycle than seabream (80%) in spite of equal mortality due to pathogen infections (10%). The differences in survival may be explained by mortality “of other causes”. Seabream and seabass have different disease profiles, and the profile is slightly different between geographical regions. Among the most important diseases, tenacibaculosis and vibriosis were identified in seabass and Sparicotyle chrysophrii (a gill fluke) and nodavirus in seabream. Correlating mortality data to management variables showed that increasing density, buying fingerlings from external sources, and treatments due to disease are factors that negatively influence mortality rate. Most of the surveyed farms did not keep sufficient quality data to implement good health status reports and perform detailed impact studies, which shows the necessity of updating the current legislative framework to provide the basis for better reporting of relevant pathogens in the Mediterranean basin.info:eu-repo/semantics/publishedVersio

A Single-Tube HNB-Based Loop-Mediated Isothermal Amplification for the Robust Detection of the Ostreid Herpesvirus 1

The Ostreid herpesvirus 1 species affects shellfish, contributing significantly to high economic losses during production. To counteract the threat related to mortality, there is a need for the development of novel point-of-care testing (POCT) that can be implemented in aquaculture production to prevent disease outbreaks. In this study, a simple, rapid and specific colorimetric loop-mediated isothermal amplification (LAMP) assay has been developed for the detection of Ostreid herpesvirus1 (OsHV-1) and its variants infecting Crassostrea gigas (C. gigas). The LAMP assay has been optimized to use hydroxynaphthol blue (HNB) for visual colorimetric distinction of positive and negative templates. The effect of an additional Tte UvrD helicase enzyme used in the reaction was also evaluated with an improved reaction time of 10 min. Additionally, this study provides a robust workflow for optimization of primers for uncultured viruses using designed target plasmid when DNA availability is limited.info:eu-repo/semantics/publishedVersio

A Production Calendar Based on Water Temperature, Spat Size, and Husbandry Practices Reduce OsHV-1 μvar Impact on Cultured Pacific Oyster Crassostrea gigas in the Ebro Delta (Catalonia), Mediterranean Coast of Spain

Since 2006, the production of Pacific oyster Crassostrea gigas in the Ebro Delta area has dramatically declined from around 800 metric tons (MT) per year to 138 MT in 2011. This decline in production has had a significant socio-economic impact in a region where the shellfish sector is a traditional economic activity for many families. The identified agent responsible for this reduction in C. gigas production was Ostreid Herpesvirus microvar (OsHV-1 μvar), which has been associated with C. gigas spat mortalities in France, and in many other countries. In Spain the episodes of mortality became critical for the regional shellfish production between 2008 until 2014, with mortality percentage up to 100%. In this study, local hatchery C. gigas spat was used as sentinel animals for epidemiological studies and management tests carried out with the aim of reducing oyster mortality in the Ebro Delta area. A production calendar mainly based on water temperature dynamics was designed around an optimal schedule for spat immersion. The mmersion calendar included two optimal periods for spat immersion, in summer when temperatures are ≥25◦C and at the end of autumn and beginning of winter when they are ≤13◦C. Such production planning has reduced mortalities from 80% (in 2014 and previous years) to 2–7.5% in 2015 in cemented oysters. Furthermore, other recommendations related to spat immersion size, culture density and methodology, and cementing calendar, which helped to achieve the results presented, were also recorded and transferred to local producers. This work presents a successfully tested management strategy reducing OsHV-1 μvar impact by designing new field management practices mainly focused on the handling and timing of spat immersion. This approach could be used as a management model in areas presenting similar production practices and environmental characteristics.info:eu-repo/semantics/publishedVersio

The Use of a DNA-Intercalating Dye for Quantitative Detection of Viable Arcobacter spp. Cells (v-qPCR) in Shellfish

The genus Arcobacter (Vandamme et al., 1991), comprised of Campylobacter-related species, are considered zoonotic emergent pathogens. The presence of Arcobacter in food products like shellfish, has an elevated incidence worldwide. In this study, we developed a specific viable quantitative PCR (v-qPCR), using the dye propidium monoazide (PMA), for quantification of the viable Arcobacter spp. cells in raw oysters and mussels. The high selectivity of primers was demonstrated by using purified DNA from 38 different species, 20 of them from the genus Arcobacter. The optimization of PMA concentration showed that 20 μM was considered as an optimal concentration that inhibits the signal from dead cells at different concentrations (OD550 from 0.2 to 0.8) and at different ratios of live: dead cells (50:50 and 90:10). The v-qPCR results from shellfish samples were compared with those obtained in parallel using several culture isolation approaches (i.e., direct plating on marine and blood agar and by post-enrichment culturing in both media). The enrichment was performed in parallel in Arcobacter-CAT broth with and without adding NaCl. Additionally, the v-qPCR results were compared to those obtained with traditional quantitative (qPCR). The v-qPCR and the qPCR resulted in c.a. 94% of positive detection of Arcobacter vs. 41% obtained by culture approaches. When examining the reduction effect resulting from the use of v-qPCR, samples pre-enriched in Arcobacter-CAT broth supplemented with 2.5% NaCl showed a higher reduction (3.27 log copies) than that of samples obtained directly and those pre-enriched in Arcobacter-CAT broth isolation (1.05 and 1.04). When the v-qPCR was applied to detect arcobacter from real shellfish samples, 15/17 samples tested positive for viable Arcobacter with 3.41 to 8.70 log copies 1g-1. This study offers a new tool for Arcobacter surveillance in seafood.info:eu-repo/semantics/publishedVersio