Author: marina02

Highlighting gill pathology – Better visualisation of an often complex picture

Gill issues in salmon, such as infectious diseases and damage from environmental stressors, significantly impact their respiratory efficiency, growth rates, and overall health, posing substantial challenges for sustainable aquaculture.

Histopathology is a powerful tool when assessing fish gills but interpretation of histopathology reports can be quite overwhelming. Histology reports are often lengthy and dense, with semi-quantitative assessment provided at best. Assessing changes between groups of fish over time or space can also be challenging.

In an increasingly visual and data heavy world, we need tools to aid interpretation of complex data and make information more approachable and appealing.

A graphical method of representing histological findings in gills, the “gill index”, was recently developed, based on semi-quantitative scoring of gill lesions. This method reflects the severity of 4 key pathological processes (inflammation, circulatory disturbance, cell damage and hyperplasia) and provides an overall “gill index” facilitating interpretation and comparison of patterns of damage between histopathology reports. Adding visual cues to histopathology reports empowers end-users to better identify the key changes and visualize their severity.

Examples of patterns of gill pathology from varying clinical scenarios will be presented along with their gill index visualization to illustrate the value of adding graphical elements to such reports.

Better understanding and presenting key drivers of gill changes will assist fish health providers in improved monitoring of gill health both during disease outbreaks and before and after routine husbandry operations.

1. Baily, Johanna, Patogen AS, Author
2. Herrero, Ana, Patogen AS, Author
3. Thoen, Even, Patogen AS, Author
4. Rodger, Hamish, Patogen AS, Author
5. Holm, Helle, Patogen AS, Author
6. Moen Hansen, Kristoffer, Patogen AS, Presenter
7. Wustner, Stefanie, Patogen AS, Author

Pacific oyster explants provide new controlled model to study OsHV-1 replication

Since the 1990s, Pacific oysters (Magallana gigas), particularly juveniles, have experienced recurrent mass mortality events. These events are strongly associated with infection by Ostreid herpesvirus type 1 (OsHV-1), a major viral pathogen that causes significant economic losses in oyster aquaculture. Despite the importance of this disease, our understanding of the mechanisms underlying viral infection and the host immune response remains limited, largely due to the lack of established marine mollusc cell lines.

Recently, the use of oyster tissue explants has emerged as a promising alternative, providing a system that mimics primary cell culture conditions.

In this study, gill explants were cultured in vitro and experimentally infected with OsHV-1. We monitored infection dynamics over 48 hours and assessed the effects of the virus on tissue structure and integrity by direct observation. Explants were also sampled at defined time points for OsHV-1 quantification by qPCR, as well as for DNA and cDNA long-read Nanopore sequencing.

Our results show that OsHV-1 can replicate in gill explants, providing a controlled system for studying viral replication dynamics. We successfully reassembled complete OsHV-1 genomes from infected explants and observed gene expression kinetics that closely mimic those reported in in vivo studies. This explant model provides a valuable approach to study host-pathogen interactions in M. gigas under controlled conditions and offers new insights into tissue-specific immune responses to OsHV-1.

1. Dotto-Maurel, Aurélie, Ifremer, Presenter
2. Sullivan, Kallen, Roslin Institute, Author
3. Morga, Benjamin, Ifremer, Author
4. Bean, Tim, Roslin Institute, Author
5. Chevignon, Germain, Ifremer, Author

Global Warming and Aquatic Vulnerability: Salmonid Mortality as an Indicator of Climate Stress

Rapid climate change is an unprecedented global challenge with far-reaching consequences for biodiversity, particularly in aquatic ecosystems. Studies have consistently demonstrated that temperature is a critical factor influencing the health, distribution, and survival of cold-water fish species. For Atlantic salmon (Salmo salar) and brown/sea trout (Salmo trutta), temperatures above approx. 20°C, and for Arctic char (Salvelinus alpinus) temperatures above 16°C, significantly stress fish, compromising their immune systems and increasing susceptibility to diseases such as bacterial infections, parasites, and fungal outbreaks.  Recent reports from Swedish fish farmers indicate shifts in fish health, such as the near disappearance of protozoan parasites, reduced efficacy of bath treatments against external infections, and an increased occurrence of fungal infections in juvenile fish. In addition, compiled data from River Umeälven shows an increasing trend for average annual temperature, days above 5°C (“growth days”) and days above 20°C since 1949. This suggests that changes in environmental conditions are influencing the pathogen-host dynamics, potentially leading to the emergence of new diseases or the re-emergence of previously controlled pathogens.

As the climate continues to warm, the environmental changes are expected to become more severe. The shift in ecological balance, coupled with an increased susceptibility to disease, underscores the urgent need for comprehensive research into the impacts of climate change on aquatic ecosystems. By understanding these dynamics, we can better predict future trends and implement adaptive management strategies to protect vulnerable fish populations by ensuring sustainable aquaculture practices. As a pilot project, we integrated long-term temperature records with mortality data from three fisheries in River Umeälven in northern Sweden to model the impact of rising water temperatures on the mortality rates of salmon , brown/sea trout, and Arctic char. Our generated model demonstrates a strong correlation between rising temperatures and fish mortality, particularly during June – August, when water temperatures peak. Moreover, the analysis identifies key protective factors, including fish size and the rate of temperature change, suggesting that both biological and environmental parameters influence temperature-driven mortality. While the model provides compelling evidence of the relationship between temperature fluctuations and fish mortality, it remains predictive and requires further validation over time to refine its accuracy and applicability to other river systems, species and inclusion of additional parameters. The final goal is to be able to predict future events.

1. Vidal, Gema, Swedish Veterinary Agency, Author
2. Johansson, Robert, Swedish Veterinary Agency, Author
3. Axén, Charlotte, Swedish Veterinary Agency, Presenter
4. Persson, David, Swedish Veterinary Agency, Author

Beyond the stain: investigating Ziehl-Neelsen negative granulomas in fish and human skin using molecular diagnostics

Introduction. Mycobacterial infection is commonly considered a potential cause of granulomatous lesions. Traditionally, mycobacteria are classified into two main groups: the Mycobacterium tuberculosis complex and non-tuberculous mycobacteria (NTM). In humans, the skin lesion called “fish tank granuloma” is typically associated with Mycobacterium marinum, an NTM, and the transmission occurs through contact with contaminated water or infected fish via skin lesions. However, increasing evidence points to Mycobacterium chelonae involvement in granulomatous infections affecting both humans and fish. Ziehl-Neelsen (ZN) staining remains a widely used standard histochemical technique for the detection of mycobacteria. Nonetheless, several studies have reported its limited sensitivity in identifying certain NTM species, including M. chelonae. Consequently, in this study, we investigated ZN-negative granulomas in adult meagres (Argyrosomus regius) and humans by combining histopathology with molecular diagnostic techniques.

Methodology. Formalin-fixed, paraffin-embedded samples from 18 livers of meagres and 13 human skin biopsies (upper limb: n=9; lower limb: n=2; back: n=1; trunk: n=1) showing ZN-negative granulomas were subjected to PCR amplification and sequencing of the 65-kDa heat shock protein (hsp65) gene. Sequence identification was performed using GenBank’s Basic Local Alignment Search Tool (BLAST), and phylogenetic analysis was conducted using ClustalW and MEGA v.7 software.

Results. Histopathological examination revealed nodular or diffuse granulomatous inflammation, characterised by numerous macrophages and epithelioid cells with scattered multinucleated giant cells, rims of lymphocytes, and plasma cells, all surrounded by spindle-shaped fibroblasts in humans, and multifocal typical granulomas without giant cells in fish. Biomolecular analysis revealed the presence of M. chelonae in 5 out of 18 meagre liver samples and 6 out of 13 human cases, all involving the upper limbs.

Conclusions. These findings suggest that M. chelonae could be negative to the ZN stain and should be considered as a potential pathogen in fish and humans, particularly in cases involving the upper limbs. Based on our preliminary results, we strongly recommend including molecular diagnostic tools in the evaluation of granulomatous lesions of unknown aetiology when histochemical methods fail to identify the causative agent. This study also highlights the One Health perspective, emphasizing the interconnection between human, animal, and environmental health in the management of zoonotic infections.

1. Cubeddu, Tiziana, Department of Veterinary Medicine, University of Sassari, Sassari, Italy, Author
2. Pilloni, Luca, Department of Medical Sciences and Public Health, University Hospital and University of Cagliari, Cagliari, Italy, Author
3. Burrai, Giovanni Pietro, Department of Veterinary Medicine, University of Sassari, Sassari, Italy, Author
4. Sanna, Marina Antonella, Department of Veterinary Medicine, University of Sassari, Sassari, Italy, Author
5. Polinas, Marta, Department of Veterinary Medicine, University of Sassari, Sassari, Italy, Author
6. Murgia, Claudio, Department of Veterinary Medicine, University of Sassari, Sassari, Italy, Author
7. Gerosa, Clara, Department of Medical Sciences and Public Health, University Hospital and University of Cagliari, Cagliari, Italy, Author
8. Ambu, Rossano, Department of Medical Sciences and Public Health, University Hospital and University of Cagliari, Cagliari, Italy, Author
9. Fanni, Daniela, Department of Medical Sciences and Public Health, University Hospital and University of Cagliari, Cagliari, Italy, Author
10. Antuofermo, Elisabetta, Department of Veterinary Medicine, University of Sassari, Sassari, Italy, Presenter

A single-nuclei atlas of whole Pacific oysters in response to OsHV-1 infection

The Pacific oyster (Magallana or Crassostrea gigas) is a globally important species in aquaculture, yet its production remains threatened by recurring outbreaks of Ostreid herpesvirus 1 microvariant (OsHV-1 µVar), which can cause mass mortalities in juvenile oysters. Despite the scale of this problem and significant economic losses, our understanding of precise host cellular responses to the virus at different stages of the infection remains limited. Here, we present the first single-nucleus RNA sequencing (snRNA-seq) analysis of whole oysters to capture the dynamic landscape of host response to OsHV-1 µVar infection over a 72-hour infection cycle. We were able to capture transcriptomic changes at single-nucleus resolution encompassing early, mid, and late stages of the infection.

Using Parse Biosciences Evercode chemistry, we sequenced ~3.2 billion reads and profiled over 23,000 nuclei from OsHV-1 µVar bath-challenged and control oysters, leading to the identification of 18 distinct transcriptional clusters, including five discrete haemocyte immune cell types. These clusters represent cells from key oyster tissues, namely gill, mantle, adductor muscle, digestive gland, hepatopancreas, and immune cell types, providing a detailed atlas of oyster cell diversity. Temporal analysis revealed stage-specific changes in gene expression, with marked transcriptional shifts in haemocytes and gill tissues across infection stages. Differential gene expression analysis revealed subset-specific activation of antiviral and apoptotic regulatory networks within haemocytes and gill subtypes, and most notably in hepatopancreas.

In addition to characterizing infection-associated transcriptomic changes, we provide a curated list of cell-type-specific marker genes for key oyster tissues and immune cells, creating a valuable resource for the bivalve research community. Our analyses also underscore the current limitations of genome annotation in M. gigas, which remains a major bottleneck for functional interpretation and pathway mapping in non-model marine invertebrates

1. DEWARI, POORAN, UNIVERSITY OF EDINBURGH, Presenter
2. Chapuis, Ambre, UNIVERSITY OF EDINBURGH, Author
3. Florea, Alexandra, UNIVERSITY OF EDINBURGH, Author
4. Furniss, James, UNIVERSITY OF EDINBURGH, Author
5. Regan, Tim, UNIVERSITY OF EDINBURGH, Author
6. Bean, Tim, UNIVERSITY OF EDINBURGH, Author

Dams and reservoirs trigger temperature-dependent disease

Dams are ubiquitous in river systems, providing essential services such as drinking water, electricity and irrigation to human society. At the same time, dams significantly impact ecosystems by disrupting flow, and altering natural water temperature regimes. Here, we describe a novel, unappreciated threat posed by dams and reservoirs to one of the world’s most popular game fish, brown trout (Salmo trutta). We show that small river impoundments elevate downstream water temperature in summer, which increase the prevalence and abundance of malacosporean parasite Tetracapsuloides bryosalmonae triggering proliferative kidney disease (PKD), an emerging disorder in salmonids across North America and Europe. Our study highlights the role of reservoirs in creating parasite and disease hotspots, while providing limited evidence that dams act as barriers to T. bryosalmonae spread. As global temperatures continue to rise, reservoirs are likely to cause unsustainable effects on downstream riverine fishes through temperature-induced diseases, with particularly severe consequences for cold-water salmonids. This makes downstream areas from reservoirs valuable sentinel sites for monitoring climate impacts on riverine ecosystems. Ultimately, the assessment of dams requires a more holistic approach, where the disease risks are included in the decision-making process balancing human needs with the health of aquatic ecosystems.

1. VASEMÄGI, ANTI, SWEDISH UNIVERSITY OF AGRICULTURAL SCIENCES, Presenter
2. Magnus, Lauringson, Estonian University of Life Sciences, Author
3. Joacim, Näslund, SWEDISH UNIVERSITY OF AGRICULTURAL SCIENCES, Author
4. Lilian, Pukk, Estonian University of Life Sciences, Author
5. Siim, Kahar, Estonian University of Life Sciences, Author
6. Riho, Gross, Estonian University of Life Sciences, Author

A CHIP TOOL FOR NON-INVASIVE DETECTION OF DISEASE-CAUSING ORGANISMS IN DANISH FISH AQUACULTURE

When clinical fish diseases are discovered in aquaculture facilities, the fish are usually already very sick. Treatment is initiated immediately, but decreased welfare, increased mortality and economic loss is, nonetheless, often a consequence. Therefore, prophylaxis and non-invasive early detection of disease-causing pathogens can facilitate interventions prior to clinical outcome and severe disease. We have developed a Fluidigm chip, tool based on environmental DNA/RNA (collectively eNA) sampled from water for early detection and surveillance of 11 pathogens relevant for Danish aquaculture. For three of the pathogens, controlled experiments have been conducted, correlating the eNA levels in the water with the disease status in the fish. The results from these experiments showed that pathogen-specific eNA of the targeted organisms was elevated in the water before clinical signs appeared in the fish. Our tool can therefore be used to detect pathogens in the early phase of infection, days before disease becomes severe, facilitating intervention before fish show clinical signs of the disease. This can motivate the farmers to intervene earlier (if possible) to limit disease and increase the welfare of the fish, improving sustainable animal production.

1. Marana, Moonika, University of Copenhagen, Presenter
2. von Gersdorff Jørgensen, Louise, University of Copenhagen, Author
3. Larsen, Helene, Technical University of Denmark, Author
4. Christiansen, Debes, The Faroese Food and Veterinary Authority, Author
5. Pedersen, Petra, The Faroese Food and Veterinary Authority, Author
6. Dahl, Maria, The Faroese Food and Veterinary Authority, Author
7. Henard, Cyril, University of Copenhagen, Author
8. Duan, Yajiao, University of Copenhagen, Author

Potential of eDNA approaches for the monitoring of marine bivalve parasites: example of the ROME project

Environmental DNA approaches are increasingly used to detect microorganisms in environmental compartments, including water. They show considerable advantages to investigate and monitor microorganisms such as bivalve parasites.

An eDNA-based monitoring network named ROME (Réseau d’Observatoires de Microbiologie Environmentale intégrée, rome.ifremer.fr) piloted by Ifremer was deployed in France between 2020 and 2024 in four estuarine ecosystems that integrate oyster farms: Baie des Veys in Normandy, Rade de Brest in Brittany, Marennes Oleron in New Aquitania, and Etang de Thau in Occittania. This network aimed at evaluating the influence of river inputs on the structuration of estuarine microbiomes and the potential emergence of new microbiological risks for human, aquaculture and ecosystem health.

In the four studied ecosystems, water was collected fortnightly at both a coastline and an off-shore sampling stations. Additionally, adult oysters were collected monthly.  Nucleic acids were extracted from water samples after filtration at 20, 3 and 0.22µm and from oyster tissues. Amplification and Illumina sequencing of the V4 and V1-V2 18S rDNA regions allowed analyzing the protistome from water samples and oysters, respectively.

Sequences of taxa belonging to parasite genera infecting bivalves were detected in water and oyster samples. The geographic origin as well as the nature of samples (coastline and off-shore water, oyster) had an influence on the taxa detected. These results show the potential of such eDNA-metabarcoding approach to detect the presence of parasites but also to better understand their distribution in coastal ecosystems.  This study also highlights the need for standardization and validation of the method before its application for routine monitoring.

1. ARZUL, ISABELLE, IFREMER, Presenter
2. LECADET, CYRIELLE, IFREMER, Author
3. CANIER, LYDIE, IFREMER, Author
4. CHEVIGNON, GERMAIN, IFREMER, Author
5. SERGHINE, JOELLE, IFREMER, Author
6. PPARNAUDEAU, SYLVAIN, IFREMER, Author
7. NOEL, CYRIL, IFREMER, Author
8. SIANO, RAFFAELE, IFREMER, Author

Validation of multi-target high-throughput qPCR for pathogen detection in salmonids

Introduction: Aquaculture production has been growing steadily worldwide since the 1990’s, and environmental restrictions to reduce pollution have pushed the development of recirculating aquaculture systems (RAS). In Denmark, this has resulted in fewer farms but with more intense production, leading to more frequent introduction of new batches of eggs or juveniles. This increases the risk of potentially introducing microbial pathogens into farms, and disease cases may turn from one pathogen = one disease to more complex diseases with multiple agents playing a role.

To ease testing of several pathogens, we have developed and validated a multi-target high-throughput microfluidic qPCR (HT-qPCR) method, which currently allows detection of 20 different pathogens and putative pathogens. The method was used in a longitudinal field study, which coincidentally occurred during a disease outbreak with increased mortality.

Methodology: Validation of the HT-qPCR method was performed in steps: 1) Testing specificity using known positive reference material, 2) testing limit of detection using artificial dsDNA and ssRNA, 3) testing using known positive tissue material, 4) testing impact of pre-amplification and exonuclease treatment, 5) comparison to current standard diagnostic methods in a field study, and 6) comparison of qPCR, dPCR and HT-qPCR (limit of detection).

In cases where it is relevant, the assays listed in the diagnostic manual (EURL for fish diseases) were used.

Results: Overall, the method is robust, and results are comparable to current diagnostic methods. Limit of detection is similar to qPCR, and pre-amplification of samples does not impact detection of a target in low concentrations in the presence of a highly concentrated target. During a longitudinal field investigation, a disease outbreak with increased mortality occurred (37% of the unit succumbed) in which Piscine orthoreovirus 3 and Candidatus Branchiomonas cysticola were detected in fish samples.

Conclusions: Multi-target HT-qPCR is a robust method which produces results comparable to current diagnostic methods. Some assays targeting bacterial pathogens did however not perform according to standard and needs revision. Furthermore, validation was performed using cell culture, bacterial cultures, and known positive tissue samples. While the method was tested using water samples from the field, the use of the method for environmental nucleic acids (eNA) needs to be validated. However, interpretation of eNA data should be done carefully, taking the production system into account. Field investigation showed that there was a constant low presence of several bacterial pathogens in the water which did not necessarily infect the fish.

1. Sørensen, Juliane, DTU Aqua, Presenter
2. Vendramin, Niccolò, DTU Aqua, Author
3. Madsen, Lone, DTU Aqua, Author
4. Cuenca, Argelia, DTU Aqua, Author

Implementation of a Recombinase Polymerase Amplification-Driven Duplex Lateral Flow Diagnostic Kit for Concurrent Detecting Infectious Pancreatic Necrosis Virus and Viral Hemorrhagic Septicemia Virus: A Comparative Analysis of RPA and PCR Methods.

Diagnosis time of infectious diseases in aquaculture is an important issue. Local and commercial enterprises generally do not prefer laboratory diagnosis of infectious diseases due to the long lead time and cost. This leads to the under-reporting of outbreaks in aquaculture farms and the recognition of infectious agents when they spread to many farm systems and cause significant economic loss. In this study, we aimed to develop a diagnostic kit that is a fast, simple and mobile tool that the breeder can easily apply under field conditions against Infectious pancreatic necrosis virus (IPNV) and Viral hemorrhagic septicemia virus (VHSV), which are two crucial viral pathogens that cause significant problems in trout farming in Türkiye, both in hatcheries and farms. For this purpose, we developed a rapid diagnosis suitcase that can be easily performed by the breeders in the field, thanks to the lateral flow test stick that we developed for the duplex diagnosis of VHSV and IPNV, based on Recombinase polymerase amplification (RPA), one of the molecular tests developed in recent years. To evaluate the sensivity of the lateral flow RT-RPA, we have also developed real-time RT-PCR and real-time RT-RPA methods which are targeted the same gene region of VHSV and IPNV. All the assays were optimized both singleplex and dubleks for both viruses. As a result of the optimizations, the minimum detection limits of all methods were determined. For singleplex detection, real-time RT-RPA is found the most sensivite assay for IPNV. For the VHSV, real-time RT-PCR and real-time RT-RPA assays are found the most sensitive assays. For the dublex detection, we found that the lateral flow RT-RPA assay can detect as well as real-time RT-PCR but not real-time RT-RPA for VHSV(Fam-labelled). For IPNV (DIG-labelled), lateral flow RT-RPA assay is ten-fold lower than real-time RT-PCR and real-time RT-RPA. We consider that detection limits for lateral flow RT-RPA are within acceptable limits for both IPNV and VHSV in dubleks detections. For this purpose, we prepared a mini-suit case containing micro-centrifuge, thermal block and pipette. So the lateral flow RT-RPA assay can be carried out under field conditions. It is thought that the mobile suitcase laboratory will allow timely diagnosis in the field and will be a pioneer for the kits to be developed with the same system infrastructure for the detection of infectious diseases of other farm animals and humans.

1. TAMER, CÜNEYT, ONDOKUZ MAYIS UNİVERSİTY, VETERINARY FACULTY, TÜRKİYE, Presenter
2. KURUÇAY, HANNE NUR, ONDOKUZ MAYIS UNİVERSİTY, VETERINARY FACULTY, TÜRKİYE, Author
3. GÖZEL, SEDA, ONDOKUZ MAYIS UNİVERSİTY, VETERINARY FACULTY, TÜRKİYE, Author
4. MUFTUOGLU, BAHADIR, ONDOKUZ MAYIS UNİVERSİTY, VETERINARY FACULTY, TÜRKİYE, Author
5. KADI, HAMZA, SAMSUN VETERINARY CONTROL INSTITUTE, TURKİYE, Author
6. DOĞAN, FATİH, SAMSUN VETERINARY CONTROL INSTITUTE, TURKİYE, Author
7. YILDIRIM, SERDAR, SAMSUN VETERINARY CONTROL INSTITUTE, TURKİYE, Author
8. GÜMÜŞOVA, SEMRA, ONDOKUZ MAYIS UNİVERSİTY, VETERINARY FACULTY, TÜRKİYE, Author
9. YAZICI, ZAFER, ONDOKUZ MAYIS UNİVERSİTY, VETERINARY FACULTY, TÜRKİYE, Author
10. WEIDMANN, MANFRED, MEDİZİNİSCHE HOCHSCHULE BRANDENBURG THEODOR FONTANE, 01968 SENFTENBERG, GERMANY, Author
11. ALBAYRAK, HARUN, ONDOKUZ MAYIS UNİVERSİTY, VETERINARY FACULTY, TÜRKİYE, Author