Author: marina02

development of point of need diagnostic methods for detection of bonamia ostreae using in-field nucleotide sequencing

Introduction

Populations of the European flat oyster (Ostrea edulis) have been declining globally due to overexploitation and diseases caused by parasites such as Bonamia ostreae. While technological advances have significantly improved the detection and identification of this pathogen in laboratory settings, there is a pressing need for diagnostic techniques for point-of-need use. Rapid diagnosis of infectious diseases in farms, border controls, and processing units might allow the implementation of measures to mitigate and prevent the spread of diseases. This is particularly crucial for diseases that cannot be eradicated or for which there are limited or no treatments, which is the case for most diseases affecting molluscs. The objective of this study was to develop diagnostic methods for in-field detection and characterization of B. ostreae using in-field nucleotide sequencing.

Methodology

Nine different DNA extraction methods were evaluated to determine their efficiency in obtaining DNA from oyster samples under field conditions. The quantity and quality of the DNA extracted was assessed using fluorescence and spectrophotometric methods. Additionally, PCR amplification of host DNA was performed to assess the presence of inhibitors. For amplification of B. ostreae DNA, fast PCR and recombinase polymerase amplification (RPA) assays were developed. The assays were designed to allow the differentiation of B. ostreae, B. exitosa and B. perspora. Finally, rapid ONT library protocols were implemented to sequence fast PCR and RPA products.

Results and Conclusions

Among the nine DNA extraction methods tested, the Quickextract™ DNA extraction solution was deemed the most suitable based on dna yield (ng/mg), 260/280 and 230/280 ratios, absence of inhibitors, extraction time, and simplicity. The fast PCR approach successfully detected B. ostreae in samples with DNA copy numbers between 245 to 6918 per mg. Using the fast PCR/ONTapproach, a consensus sequence of approximately 500 bp with 100% nucleotide similarity to the reference B. ostreae was obtained. This method enabled the detection and characterization of B. ostreae from an infected oyster in approximately 2 hours (from tissue to sequence). However, this approach tested only a single sample, unlike the RPA with SYBR™ green method, which allowed simultaneous testing of up to 12 samples. RPA products were also successfully sequenced using the ONT rapid barcoding kit. Analysis of naturally infected O. edulis indicated that RPA and fast PCR have similar sensitivity levels.

Keywords

point of need testing, dna extraction, rpa, fast pcr, in-field sequencing, nanopore sequencing, Bonamia ostreae

Funding

FC1216 (DEFRA) and Cefas Seedcorn dp445

1. Batista, Frederico, Cefas, Presenter
2. Kerr, Rose, Cefas, Author
3. Edwards, Matt, Cefas, Author
4. Robert, Hatfield, Cefas, Author
5. Scott, George, Cefas, Author

Mussel (Mytilus edulis) mortality events in association with Francisella halioticida in the Netherlands

Mass mortality events of mussels have been reported in the Netherlands as early as 1949, when the introduction of Mytilicola intestinales was attributed to a massive decline in the mussel population. Other mortality events have been attributed to anoxic conditions created by spring blooms of Phaeosystis globosa in 2001. However, since 2016 a series of mussel mortalities have taken place in the Oosterschelde area (NL) without a clear cause of the mortality. In all cases a high incidence of granuloma and other hemocytic lesions have been observed by histology. Retrospective histological analysis of mortality and non-mortality batches showed that the presence of granuloma’s or granuloma-like structures seems to increase over the recent years but not restricted to mortalities.

Granuloma’s have been associated with the presence of the bacterium Francisella halioticida for several mollusc species. Using a real time PCR assay for the detection of F. halioticida, the bacterium could be detected in the mussel samples from the Oosterschelde. The presence of F. halioticida, seems to be associated with mortalities, however there is no clear relationship between the presence of the hemocytic lesions and F. halioticida.

A multidisciplinary study involving scientists, mussel farmers and policymakers has been initiated in 2025 with the aim to better understand the mussel mortalities in the Oosterschelde: the causes, trends and risk factors.

1. Engelsma, Marc, WBVR, Presenter
2. Voorbergen-Laarman, Michal, WBVR, Author
3. Van Gelderen, Betty, WBVR, Author
4. Bouras, Helene, UniCaen, Author
5. Schotanus, Jildou, WMR, Author
6. Capelle, Jacob, WMR, Author

Spatiotemporal detection of Aphanomyces astaci after crayfish plague outbreaks

Introduction:

The oomycete Aphanomyces astaci is the causative agent of crayfish plague in native European crayfish, leading to high mortality. Invasive North American crayfish serve as carriers of the agent. Disease dynamics and spread monitoring in natural water systems remains a major challenge, as outbreaks often occur rapidly and unpredictably.

In this context, environmental DNA (eDNA) has gained attention as non-invasive method to trace the presence of aquatic pathogens, including A. astaci, in water bodies making it particularly valuable for post-outbreak surveillance and early warning systems.

Our aims were to characterise disease dynamics in natural waters following crayfish plague outbreaks by 1) monitoring crayfish populations and 2) detecting spatio-temporal trends of A. astaci eDNA detection.

Methodology:

In this study we explore the integration of aquatic eDNA analysis with crayfish population monitoring to assess the spread of A. astaci following a crayfish plague outbreak.

We used a field-based sampling method with Sterivex™ filter cartridge followed by qPCR detection of A. astaci DNA in different water bodies in Switzerland after crayfish plague outbreaks. Where possible, monitoring of crayfish populations was performed to check for crayfish occurrence, mortality and presence of pathogen in crayfish tissue. A. astaci genotype groups involved were determined through qPCR.

Four water bodies, one pond and three rivers, were monitored over several years with varying sampling frequencies. In the rivers, multiple sampling sites were established both upstream and downstream of the outbreak location. Water samples were collected and filtered directly in the field, and the filters were subsequently analyzed for the presence of A. astaci DNA.

Results:

Outbreaks were caused by A. astaci genotypes B, D and E. Remarkably, A. astaci eDNA was detected for more than a year after the initial outbreak, indicating either ongoing, undetected transmission of the pathogen or prolonged persistence of its DNA in the environment.

Conclusions:

A. astaci eDNA detection alone cannot distinguish between a current presence of A. astaci or rather delayed picture of past disease dynamics in the water body. The solely eDNA detection can be used to detect past or current presence of A. astaci in a water body. Interpreting these findings remains complex and requires a thorough understanding of various influencing factors, including the ecology of crayfish plague, the behaviour and degradation of eDNA, environmental conditions such as water body characteristics and season, and potential human influences.

1. Pisano, Simone Roberto Rolando, FIWI – Institute for Fish and Wildlife Health, Presenter
2. Steiner, Jonas, FIWI – Institute for Fish and Wildlife Health, Author
3. Jemmi, Eliane, FIWI – Institute for Fish and Wildlife Health, Author
4. Zürcher, Manon, FIWI – Institute for Fish and Wildlife Health, Author
5. Zingre, Tatiana, FIWI – Institute for Fish and Wildlife Health, Author
6. Kreienbühl, Thomas, Ecqua GmbH, Fisch- und Gewässerökologie, Author
7. Randegger, Florian, Departement Bau, Verkehr und Umwelt, Abteilung Wald, Jagd und Fischerei, Aarau, Author
8. Kugler, Michael, Amt für Natur, Jagd und Fischerei, Davidstrasse 35, St. Gallen, Author
9. Schmidt-Posthaus, Heike, FIWI – Institute for Fish and Wildlife Health, Author

STRATEGIES TO REDUCE VIBRIO CONTENT IN THE GUT OF PACIFIC WHITE SHRIMP (Litopenaeus vannamei)

Introduction: Shrimp farming plays a vital role in global aquaculture, but it faces growing threats from bacterial diseases, particularly those caused by Vibriospecies. One of the most serious challenges is acute hepatopancreatic necrosis disease (AHPND), triggered byVibrio parahaemolyticus. As concerns over antibiotic use rise due to environmental and public health implications, alternative disease control strategies are urgently needed. This study investigated the use of fatty acids as feed additives to reduceVibriolevels in the gut of Pacific white shrimp (Litopenaeus vannamei) and to enhance their survival when challenged withVibrio parahaemolyticus.

Methodology: The study first evaluated a blend of short- and medium-chain fatty acids (capric, caprylic, butyric, and lauric acids) applied as a feed additive at inclusion levels of 0.2% and 0.4%, comparing their effects onVibrioproliferation to those of a standard reference diet. Sixty shrimp were randomly distributed into three tanks (150 liters each) and fed the respective diets over five days. Gut and fecal samples were collected and analyzed forVibriocounts using Thiosulfate-Citrate-Bile Salts-Sucrose (TCBS) agar. In a subsequent trial, 20 juvenile shrimp were challenged withVibrio parahaemolyticusTW01 at a concentration of 10⁷ CFU/mL to evaluate the protective effect of the fatty acid supplemented diets.

Results: In the gut, the 0.4% fatty acid supplemented diet resulted in significantly lower Vibrio spp. levels (7.16 × 10⁴ CFU/g) than both the reference diet (2.80 × 10⁵ CFU/g) and the 0.2% diet (1.54 × 10⁵ CFU/g) (P < 0.05), as depicted in Figure 1. During the V. parahaemolyticus challenge, shrimp fed the 0.4% fatty acid diet exhibited the highest survival rate (40%), compared to 23% for the 0.2% diet and just 7% for the reference diet (see Figure 2).

Conclusion: This study demonstrated that dietary supplementation with a 0.4% blend of short- and medium-chain fatty acids significantly reducedVibriolevels in the gut of Pacific white shrimp and improved survival duringVibrio parahaemolyticusinfection. Although growth performance was unaffected, the findings supported the potential of fatty acid-based additives as an effective, antibiotic-free approach to lowering bacterial load in shrimp aquaculture.

Keywords:Litopenaeus vannamei,Vibrio spp., feeding regimes, fatty acid additives, aquaculture

1. Birikorang, Harriet Nketiah, Former MSc. student, Laboratory of Aquaculture and Artemia Reference Center, Ghent University, Belgium, Presenter
2. Declercq, Annelies, Laboratory of Aquaculture and Artemia Reference Center, Ghent University, Belgium, Author
3. De Muylder, Eric, CreveTec BV, Ternat Belgium, Author

Phylogenomic evidence for host specialization and genetic divergence in OsHV-1 infecting Magallana gigas and Ostrea edulis

Cross-species transmission is one of the most significant causes of disease emergence in humans and other species. The Ostreid Herpesvirus type 1 has mostly been detected in the Pacific oyster Magallana gigas in the context of mortality events. However, it has been detected in other mollusk species including the flat oyster Ostrea edulis which raises questions about OsHV-1 host specialization. This study looked at the genetic differentiation of OsHV-1 in Pacific and European flat oysters, and the mechanisms that might underlie this genetic differentiation. To this end, high-throughput deep sequencing was used to analyze forty OsHV-1 genomes to characterize the genetic diversity and isolation of OsHV-1 lineages from both O. edulis and M. gigas in France as well as to assess the frequency of the virus cross-species transmission. To achieve this, comparative and population genomic, phylogenetic, and phylodynamic approaches were employed. Our findings highlight the significant influence of host species on the genetic diversity and differentiation among OsHV-1. The results suggest that the emergence of OsHV-1 in France can be traced back to the introduction of M. gigas, with the virus subsequently evolving into two distinct lineages following a cross-species transmission event. Moreover, our analysis revealed selection signals in regions responsible for crucial biological functions and domains, such as viral particle binding to host cells, DNA synthesis, replication and packaging, as well as trans- and membrane proteins, indicating the potential for OsHV-1 to adapt and specialize in response to its respective host species. To get further insights into evolutionary processes and host-virus interactions, future research should focus on investigating the coevolution between OsHV-1 and a wider range of host species using phylogenetic approaches.

1. MORGA, BENJAMIN, IFREMER, Presenter
2. PELLETIER, CAMILLE, IFREMER, Author
3. CHEVIGNON, GERMAIN, IFREMER, Author
4. FAURY, NICOLE, IFREMER, Author
5. ARZUL, ISABELLE, IFREMER, Author
6. GARCIA, CELINE, IFREMER, Author
7. CHOLLET, BRUNO, IFREMER, Author
8. RENAULT, TRISTAN, IFREMER, Author
9. JACQUOT, MAUDE, IFREMER, Author

Exploring experimentally the impact of climate change and plastic pollution on the dynamics of bonamiosis in the native European oyster Ostrea edulis

The flat oyster, Ostrea edulis, is native from Europe. Overfishing and diseases have contributed to the decline of both farmed and wild populations of this species since the 19th century. The protozoan parasite Bonamia ostreae is the etiological agent of bonamiosis and is considered as one of the main threats for the ongoing flat oyster restoration programmes occurring in different European countries. However, the 21st century also faces climate change and ever-increasing plastic pollution, which might affect the dynamics of diseases such as bonamiosis.

The vulnerability of Ostrea edulis and of its associated communities including parasites to plastic pollution in the context of climate change was investigated experimentally in the frame of the MicroCO2sm project. A 9-months mesocosm experiment simulating actual and end-of-century environmental scenarios (ocean warming, acidification and plastics) was carried out in the Oceanolab structure (Oceanopolis center for scientific and technical culture). The project aimed at assessing changes in oyster life-history traits and ecosystem structure. As flat oysters originated from a Bonamia ostreae endemic zone, the dynamics of the parasite was investigated by quantifying the parasite by Real-Time PCR in oysters at the beginning and end of the experiments as well as in dead oysters collected throughout the experiment. In addition, the presence of the parasite was tested monthly in water and sediment in order to evaluate the amount of parasites released from oysters.

Results show that regardless of the tested conditions, Bonamia ostreae can infect, develop in oysters and be released in water. While mortality associated with the parasite appeared higher in conditions involving warming and acidification, the presence of plastics seemed to favor new infections. This experimental study enabled exploring for the first time the impact of climate and pollution changes on the dynamics of bonamiosis and allowed raising hypotheses that would deserve being further investigated to support the protection and ecological restoration of Ostrea edulis.

1. ARZUL, ISABELLE, IFREMER, Presenter
2. LECADET, CYRIELLE, IFREMER, Author
3. CHOLLET, BRUNO, IFREMER, Author
4. POUSSE, EMILIEN, OCEANOPOLIS, Author
5. CASTREC, JUSTINE, IUEM, Author
6. HUVET, ARNAUD, IFREMER, Author
7. PAUL-PONT, IKA, IUEM, Author
8. DI POI, CAROLE, IFREMER, Author

Chasing the plague, one passive filter at a time

Environmental DNA (eDNA) is a rapidly advancing, powerful sampling tool that enables the detection of a species of interest and holds immense potential as a tool for large-scale disease monitoring programs in both freshwater and marine environments. It provides a non-invasive alternative to traditional field surveys and sampling methods, whereby water samples are collected as opposed to organism sacrifice. While this sampling approach is potentially superior to conventional sampling methods, it still requires substantial manpower when used as active sampling. eDNA sampling can sometimes be limited by the turbidity of water, i.e. clogging of filters limits the amount of water that can be filtered, leading to false negative results. To overcome these limitations, the current study aimed to trial in-house designed units for passive eDNA sampling fitted with three different types of filter membranes with different pore sizes.

Following a rescue effort of White-clawed crayfish (WCC) in the Awbeg River, Munster Blackwater catchment, passive sampling units were deployed in 3 sites upstream of the crayfish plague front, to determine the spread of the pathogen upstream. Three different filter types (GF, MCE 0.45µm and MCE 0.22µm) were fitted into the passive sampling unit and changed weekly. The results from these units were compared to the results from collection of 3 x 5L water samples by active filtering conducted once a month. DNA was extracted from all sample types and tested for the presence of WCC and Aphanomyces astaci, the causative agent of crayfish plague, using species specific real-time PCR assay in duplex reaction.

The results of this study demonstrate a high level of concordance between the passive and active samplers. All three membrane types have proven suitable for use with passive sampling. While active filtering of water has proven to be a reliable method for detection of crayfish and crayfish plague and has formed the basis for the National Surveillance Programme in Ireland since 2018, passive sampling being less labour intensive, would allow for larger scale, long term monitoring programmes. The preliminary results from the passive samplers, particularly when deployed for extended periods of time and using a combination of filters, provide a reliable, potentially more efficient means of tracking disease spread within infected catchments.

The use of this method helped management decisions in relation to further rescue efforts in the affected population of White-clawed crayfish in the River Awbeg.

1. Griffin, Bogna, MARINE INSTITUTE, Presenter
2. Cheslett, Deborah, MARINE INSTITUTE, Author
3. Henderson, Mox, MARINE INSTITUTE, Author

A holistic approach to bivalve health: untangling complex interactions influencing the health status of Mytilus mussel populations

Introduction: Bivalve molluscs (soft-bodied aquatic animals with two shell valves, including mussels, cockles, clams, scallops and oysters) are vital ecosystem engineers in aquatic environments, as well as some species being significant aquaculture and fisheries commodities globally. In addition to this, widely distributed, abundant coastal species like mussels are frequently utilised in biological effects monitoring programmes for environmental contaminants. Globally many mussel populations are threatened, with declines, mortalities and significant range shifts reported, particularly in populations across the North Atlantic region. Little is known about the factors driving these observed effects, and few mortality events are sufficiently characterised to identify the mechanisms behind them. It is increasingly acknowledged that these events are likely to be the result of the action of multiple factors (including pathogens, microbiomes, chemicals, food availability and environmental factors), though the identity, combination and nature of the interactions of these factors and their effect(s) on bivalve health status are not well understood.

Methodology: using the Mytilus edulis complex as a case study, we examine the known effects of environmental stressors and chemical contaminants (including pharmaceuticals, heavy metals, chemicals and microplastics) on mussels across their life cycles, and explore biomarkers currently widely utilised and recommended for use in bivalve species for biological effects monitoring of contaminants in aquatic environments. We also deploy a suite of methodologies – including established and promising approaches – to mussels from populations in the United Kingdom known to be experiencing progressive declines.

Results: We identify gaps in our understanding of the effects of anthropogenic contaminants and environmental stressors on mussels at subcellular, tissue, individual and population level, particularly at early life cycle stages including larval stages, recruitment, and settlement. We also identify shortcomings in approaches to standardised collection, storage and availability of organismal and environmental data which is likely to be informative for characterising the stressors, contaminants, and combinations thereof, which are likely to impact bivalve populations and other aquatic taxa on localised and broader scales.

Conclusions: We propose working towards a collaborative, multidisciplinary, integrated toolkit for investigation of non-infectious diseases in bivalves, and those involving both infectious agents and other causes, which is currently lacking.

1. WARD, GEORGIA, Cefas, Presenter
2. Tidy, Anna, Cefas, Author
3. White, Peter, Cefas, Author
4. Jones, Charlie, Cefas, Author
5. Bignell, John, Cefas, Author
6. Maskrey, Benjamin, Cefas, Author
7. Jessop, Ron, Eastern Inshore Fisheries and Conservation Authority, Author
8. Hooper, Chantelle, Cefas, Author
9. Bass, David, Cefas, Author

First detection of Perkinsus mediterraneus in flat oysters in Northern Adriatic Sea

Introduction

Health monitoring of Ostrea edulis in the Northern Adriatic Sea was conducted as a part of the INTERREG project “MARINET,” which aims to evaluate the feasibility of an Integrated Multi-Trophic Aquaculture (IMTA) model involving flat oysters and sea bass. To support this objective, juvenile flat oysters from Lim Bay (Croatia) were placed in lantern nets near sea bass farms at two different sites: Duino (Italy) and Budava (Croatia). Regular health checks were conducted for endemic diseases (Bonamia spp., Marteilia refringens) and other potential pathogens on both experimental and Lim Bay as native site. Oysters were sampled monthly or bimonthly over a one-year period and analyzed following EURL protocols (IFREMER).

Methodology

Histological analysis revealed a widespread presence of parasites attributable to Perkinsus mediterraneus in oyster samples from Duino, Lim Bay, and Budava. The species was confirmed by molecular analysis (PCR and sequencing), following the method described by Casas, Villalba & Reece (2002). Trophozoites and multicellular stages of P. mediterraneus were observed in the connective tissue of the visceral mass, gonads, gills, labial palps, and mantle, often associated with significant haemocyte infiltration. The prevalence of P. mediterraneus appeared to be temperature-dependent with the highest prevalence observed in autumn (October, 17.5°C), declining as the water temperatures decreased.

Results

In addition to P. mediterraneus, Bonamia exitiosa was detected from June to October but was absent in Italian site from December to February, when temperatures dropped below 15°C. A co-infection with B. exitiosa and P. mediterraneus was identified in one specimen from Duino. Furthermore, three oysters sampled in Duino exhibited disseminated neoplasia. Neoplastic cells, characterized by reduced cytoplasm, enlarged nuclei, and the presence of mitotic figures, were observed in the connective tissues of various organs and in the haemolymph.

Conclusions

The detection of Perkinsus mediterraneus, Bonamia exitiosa and disseminated neoplasia in farmed Ostrea edulis highlights the need for continued health monitoring. This is the first report of P. mediterraneus in the Northern Adriatic, with temperature appearing to influence parasite prevalence.

1. Vetri, Alessia, Istituto Zooprofilattico Sperimentale delle Venezie, ITALY, Presenter
2. Zrnčić, Snježana, Croatian Veterinary Institute, CROATIA, Author
3. D’Onofrio, Caterina, Istituto Zooprofilattico Sperimentale delle Venezie, ITALY, Author
4. Zupicic, Ivana Giovanna, Croatian Veterinary Institute, CROATIA, Author
5. Arcangeli, Giuseppe, Istituto Zooprofilattico Sperimentale delle Venezie, ITALY, Author
6. Grbin, Dorotea, Croatian Veterinary Institute, CROATIA, Author
7. Pretto, Tobia, Istituto Zooprofilattico Sperimentale delle Venezie, ITALY, Author

Preliminary analysis for the identification and localization of biomarkers of health and environmental stress in flat oyster (Ostrea edulis)

The flat oyster (Ostrea edulis) is a bivalve mollusk (Ostreidae family) native to Europe. Historically, its distribution area consisted of a wide coastal range from Norway to Morocco in the Atlantic, the entire Mediterranean basin, and the Black Sea. However, several factors, including the excessive exploitation of the resource, the deterioration of habitats, and the intensification of mortality episodes due to pathogens, have led to a progressive decline in wild and farmed populations. To date, several studies and research projects have been finalized to restore this resource, both for its importance in terms of biodiversity and for its high economic value.

Within the Interreg Europe Italy-Croatia “MARINET” project the aim is to verify the feasibility of flat oyster farming in Italy and Croatia through the use of classic and novel technologies, testing different farming protocols in various production sites. The present investigation represents a preliminary approach to monitor the health status and response to environmental stress of Ostrea edulis through immunohistochemistry (IHC).

For the implementation of the research, flat oysters were sampled from different production sites, fixed in Davidson and Bouin solutions, and routinely processed for histology and histochemistry. Furthermore, each sample was also tested by IHC to detect and localize selected biomarkers indicating a response to thermal stress as Heath shock protein 70; response to oxidative stress as Nitrotyrosine, 4-hydroxynonenal, and Malondialdehyde; response to xenobiotics such as cytochrome P450 1A (CYP1A); inflammation such as inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX-2). Additionally, indicators of apoptosis, such as Caspase-3, and antimicrobial activity, such as Lysozyme were also tested.

IHC assays showed positive results for some of the biomarkers tested. Particularly, immunopositivity was revealed for the those targeting the response to inflammation (COX-2 and iNOS), the apoptosis (Caspase-3) and the response to xenobiotics (CYP1A) in some oyster tissues (gills, digestive gland and digestive system).

These results, although still preliminary and requiring further validation with Western blotting tests, may serve as a knowledge base for the monitoring of the integrity of wild/farmed flat oyster populations and for improving farming techniques, in order to ensure the animals well-being and to increase productivity.

1. ERRANI, FRANCESCA, University of Udine, Department of Agricultural, Food, Environmental and Animal Sciences, Udine, Italy, Presenter
2. Volpatti, Donatella, University of Udine, Department of Agricultural, Food, Environmental and Animal Sciences, Udine, Italy, Author
3. Vetri, Alessia, Istituto Zooprofilattico Sperimentale delle Venezie, Legnaro (PD), Italy, Author
4. Galeotti, Marco, University of Udine, Department of Agricultural, Food, Environmental and Animal Sciences, Udine, Italy, Author