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

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

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

Presence of Vibrio mediterranei associated to major mortality in stabled individuals of Pinna nobilis L.

A major epizootic event attributed to Haplosporidium pinnae leading to 100% mortality of Pinna nobilis L. populations along Mediterranean coastlines started in the fall of 2016. As a result, a project to rescue 221 adult individuals of the endangered pen shell, Pinna nobilis was conducted in November 2017 in the two areas of the Spanish coast where the species was still abundant and apparently free from infection by H. pinnae: Port Lligat in the Costa Brava, and the Alfacs Bay in the Ebro Delta. For biosecurity reasons, the 106 individuals from the Ebro Delta were stabled at the IRTA facilities located next to Alfacs Bay, whereas the 115 individuals from Port Lligat were stabled in different institutions throughout the Spanish territory. Initial biopsies showed that individuals from the Ebro Delta were free of the parasite, whereas most individuals from Port Lligat were already parasitized and died in the following months. Individuals at IRTA were hold in five tanks and fed ca. 4% of their dry weight with a mix of three species of phytoplankton and fine riverine sediments (13% OM). Seawater was filtered through 10, 5 and 1 μm to ensure the absence of the parasite and disinfected with UV light. No individuals died during the 4 initial months of captivity, but two died in April–May at temperatures from 17 to 19 °C. A peak of mortalities occurred during the summer months and early fall (53%) with maximums coinciding with temperatures above 25 °C. Individuals were again analyzed by PCR and histology for the presence of H. pinnae, Mycobacteria sp., and other locally important pathogens of commercial bivalves (Vibrio splendidus, V. aestuarianus and Herpesvirus OsHV-1 microVar), and therefore considered as potential pathogens of pen shells. However, with the exception of 3 individuals that were positive for Mycobacteria sp., results were all negative for the studied pathogens. Microbiological culture and isolation of bacteria from three moribund individuals, sacrificed for study purposes, showed V. mediterranei as the dominant species, and further PCR analyses confirmed the presence of the bacterium in ten deceased individuals. Overall, our results suggest the V. mediterranei is an opportunistic pathogen of stabled individuals possibly subjected to stress from captivity, and that antibiotic treatment (Florfenicol) combined with vitamins and mineral supplementation and reduction of water temperature (15 to 18 °C), can be used to mitigate (not to eradicate) the disease. Further research is needed to determine diets and stabling conditions that minimize captivity stress and prevent the emergence of the disease.info:eu-repo/semantics/acceptedVersio

A Bioactive Extract Rich in Triterpenic Acid and Polyphenols from Olea europaea Promotes Systemic Immunity and Protects Atlantic Salmon Smolts Against Furunculosis

In the present study, the modulation of the transcriptional immune response (microarray analysis) in the head kidney (HK) of the anadromous fish Atlantic salmon (Salmo salar) fed a diet supplemented with an olive fruit extract (AQUOLIVE®) was evaluated. At the end of the trial (133 days), in order to investigate the immunomodulatory properties of the phytogenic tested against a bacterial infection, an in vivo challenge with Aeromonas salmonicida was performed. A total number of 1,027 differentially expressed genes (DEGs) (805 up- and 222 downregulated) were found when comparing the transcriptomic profiling of the HK from fish fed the control and AQUOLIVE® diets. The HK transcripteractome revealed an expression profile that mainly favored biological processes related to immunity. Particularly, the signaling of i-kappa B kinase/NF-kappa and the activation of leukocytes, such as granulocytes and neutrophils degranulation, were suggested to be the primary actors of the innate immune response promoted by the tested functional feed additive in the HK. Moreover, the bacterial challenge with A. salmonicida that lasted 12 days showed that the cumulative survival was higher in fish fed the AQUOLIVE® diet (96.9 ± 6.4%) than the control group (60.7 ± 13.5%). These results indicate that the dietary supplementation of AQUOLIVE® at the level of 0.15% enhanced the systemic immune response and reduced the A. salmonicida cumulative mortality in Atlantic salmon smolts.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

Use of anionic polymer-coated magnetic beads to pre-concentrate Ostreid Herpesvirus 1 from seawater: Application to a UV disinfection treatment

Ostreid Herpesvirus 1 (OsHV-1) represents a serious threat to shellfish aquaculture worldwide. To minimise its impact, early warning systems able to detect the virus in seawater prior to infection of oysters are of utmost importance. However, monitoring OsHV-1 in seawater is challenging because of its presence at very low concentrations. Thus, a rapid and simple method to pre-concentrate the virus is needed to enable detection. Herein, magnetic beads (MBs) coated with an anionic polymer were used to pre-concentrate OsHV-1 from biological matrices including oyster homogenate and seawater samples. Following virus capture, OsHV-1 DNA detection was performed by quantitative PCR (qPCR). The MB-based approach combined with qPCR attained a limit of detection (LOD) as low as 0.1 viral copy/μL, which was 100 times lower than that of the qPCR alone. This approach was applied to the analysis of OsHV-1 in seawater from an Enhanced Hydro-Optic UV (HOD-UV) disinfection experiment operating at a UV dose of 1360 J/m2 in an open flow system. Our approach enabled detection of the virus in non-treated seawater and not in UV-treated seawater, discrimination that was not possible using qPCR alone. Moreover, the strategy provided data on the pattern of kinetics of the release of the virus in seawater. The approach could find applications in shellfish hatcheries and depuration plants to ensure biosecurity requirements.info:eu-repo/semantics/acceptedVersio