Author: marina02

Cross-national genomic surveillance of viral pathogens to support disease control in Atlantic salmon aquaculture

Introduction

Viral disease outbreaks threaten the sustainability of salmonid aquaculture, resulting in economic losses and strong concerns for fish welfare. Coupling viral whole genome sequencing with epidemiological approaches can inform on disease transmission pathways, which may support control measures. This project aims to conduct viral genomic surveillance to characterise the genetic diversity of two major viruses – salmonid alphavirus (SAV) and infectious pancreatic necrosis virus (IPNV) – in Scottish and Norwegian aquaculture.

Methodology

Oxford Nanopore amplicon sequencing assays were developed to obtain viral whole genomes. Sequenced samples, and their associated epidemiological metadata, were combined with publicly available sequences to construct phylogenetic and time-calibrated phylogeographic analyses.

Results

SAV is widespread in Norwegian aquaculture, with SAV2 and SAV3 subtypes present in discrete endemic zones. Of 161 SAV samples sequenced in my study, 144 belong to the SAV3 genotype. Analyses shows four well supported clades with strong regional geographic signal within Norway. Discrete trait analyses show all clades have a strong probabilities of having an ancestral location within the Hordaland region. This provides evidence that high-density salmon farming in Hordaland acts as a primary source of SAV3 with periodic transmission events from this region responsible for outbreaks in neighbouring counties.

There have been recent reports of IPNV outbreaks in salmon genetically resistant to IPNV, with a novel sub-genotype (NG5) as the disease agent. 147 IPNV samples have been sequenced, with 104 belonging to the derived NG5 genotype and 16 the ancestral “classic” G5 (CG5). The NG5 samples show multiple signature amino acid substitutions in the capsid protein compared to CG5. Furthermore, there is an epidemiological link between contemporary NG5 circulating in Norway/Scotland and IPNV in freshwater aquaculture species circulating in Italy between 2010-2015. While there is a loose geographical relationship with sequence relatedness, a number of highly related IPNV sequences are present in both land and sea sites in geographically distant outbreaks. This suggests vertical transmission, through movement of infected ova, may play a role in IPNV spread. Additionally, 27 samples showed significant levels of mapping to both NG5 and CG5 representing potential co-infections.

Conclusions

These results highlight the value of viral genomic surveillance within aquaculture to identify the emergence of novel variants and their distribution and transmission across aquaculture sites. Such data will support disease control efforts through changes of farming practices to reduce transmission and by providing data required to produce vaccines tailored to specific pathogen strains.

1. Knight, Bertie, University of Edinburgh, Presenter
2. Eve, Oliver, Wobble Genomics, Author
3. Gundappa, Manu, Wageningen University, Author
4. Solhiem, Mari, Pharmaq Analytiq, Author
5. Matthews, Chris, Pharmaq Analytiq UK and Ireland, Author
6. Hjelle, Elise, Pharmaq Analytiq, Author
7. Alexandersen, Svein, Pharmaq, Author
8. Sandrø, Ane, Pharmaq, Author
9. Karlsen, Marius, Pharmaq, Author
10. Macqueen, Daniel, University of Edinburgh, Author

In silico characterization of the T cell receptor beta genes and locus structure in gilthead seabream

The adaptive immune response induced against pathogens or after vaccination is based on the clonal expansion of B and T lymphocytes, which express antigen-specific receptors, immunoglobulins and T cell receptors (TR), respectively. In farmed fish, the lack of specific reagents hinders the research on B/T cell mediated immune response. In human and mice, high throughput sequencing (HTS) methods have become critical tools for studying the B/T cell repertoires in many different contexts, including infection and vaccination. Conventional T helper and T cytotoxic cells express TRa/b receptors, and their diversity is mainly achieved through the somatic recombination of V/D/J segments generating the large sequence diversity required for antigen recognition. The TR loci embedding these genes are complex and poorly characterized in most teleost species. However, it is a fundamental prerequisite for accurate annotation of TR repertoire sequencing datasets and to implement HTS tools to monitor the T cell mediated immune response in farmed fish. The aim of the present work was to undertake a full annotation of the TRB locus in gilthead seabream (Sparus aurata) to increase our toolbox for immunological evaluations in this relevant aquaculture species. We annotated the high-quality gilthead seabream genome assembly (fSpaAur1.1), where the TRB locus spans 540 Kbp on the chromosome 16. A total of 412 TRBV, 3 TRBD, 62 TRBJ and 3 TRBC genes distributed in three TRB copies were identified. Each TRB copy has a TRBC gene consisting of four exons. The first TRB copy is the shortest, with 35 V genes (29 functional (F)), one D, 6 J genes and. The second copy encodes 70 V genes (59F), one D and 6 J genes. The third copy is the largest and more complex, encoding 307 V genes (211F) and 50 J genes. Interestingly, 148 of these V genes are located in reverse orientation. All functional TRBV genes can be clustered in 11 subgroups based on the nucleotide sequence identity. As found in other teleost, the seabream TRBC genes belong to 2 groups. While the first and second TRBC copy are almost identical (only one amino acid different), the third copy presents only 46% of identity with the other two. Further work is required to unveil if they have different functional roles. With the annotation of this locus in gilthead seabream we now have a key tool to study T lymphocyte dynamics and diversity, relevant, for example, in vaccine validation and evaluation.

1. PIAZZON, CARLA, IATS, CSIC, Presenter
2. Magadán, Susana, CINBIO, Author

Creating a primary cell platform from CRISPR/Cas edited Atlantic salmon (Salmo salar) alevin tissue

Introduction

Salmonids are the third most farmed fin fish species. And while the industry keeps growing, a variety of different pathogens continue to threaten its sustainability. Gene manipulation is quickly becoming a reliable way of studying gene function and disease resistance in salmon, especially when dealing with viral diseases. Methods of vector delivery into salmonid embryos, such as CRISPR/Cas9, are now well established, but the means to test the resulting embryo’s disease resistance currently relies on live pathogen exposures in whole animals, which are logistically complex and expensive, and often raise animal welfare challenges. This can be somewhat overcome by producing primary cell culture from edited animals and exposing these cultures to pathogens. However, the techniques for doing this are either not well characterized or well represented in the literature.

Atlantic salmon lags behind other salmonid species when it comes to cell cultures established from embryos or post-hatch salmon (commonly known as alevins). In this study, we have developed a protocol to culture primary cells from Atlantic salmon alevins, which allows for the testing of in-vivo gene edited cells in an in-vitro environment, reducing the need to expose whole fish to disease

Methodology:

Alevins were sacrificed, dechorionated and decontaminated using an antibiotic mix. The head tissue was chemically dissociated using trypsin for 1 hour and seeded into cell culture plates using a novel medium formulation. The cells were kept in an incubator at 20°C. A partial medium change was performed every 2-4 days for the first two weeks, after which the cells were passaged every week.

Results:

Our alevin cell cultures have similar or better survival rates when compared to other salmonid species embryonal or pre-first feed fish cell cultures. The cells, which are able to form a monolayer in just 5-7 days after seeding, remain viable and proliferative beyond two months (ongoing experiment).

Conclusions:

This new protocol for establishing primary cell cultures from hatched Atlantic salmon embryos will be a valuable tool for studying disease resistance in microinjected Atlantic salmon, reducing some of the requirements for in-vivo whole animals pathogen challenges and reducing the welfare implications.

1. Florea, Alexandra, The Roslin Institute, Presenter
2. Robledo, Diego, The Roslin Institute, Author
3. Bean, Tim, The Roslin Institute, Author

Ancient Expansion of Teleost Fc Receptor-Like Genes Diversifies Antibody Effector Functions in Early Vertebrates

Introduction

The vertebrate adaptive immune system has been shaped by a fascinating interplay between immunoglobulins and their receptors. At the heart of this defense network are Polymeric-Ig receptors (pIgRs), fragment crystallizable (FcR) receptors, and their enigmatic cousins, the Fc receptor-like molecules (FcRL). While mammals have revealed much about these receptors, their role in teleosts remains one of the most intriguing and unsolved mysteries in the vertebrate immune system.

Methodology

We combined comparative genomics, evolutionary analysis, and transcriptional profiling to explore the FcRL gene family in rainbow trout, a key teleost model. By diving deep into these receptors within the teleost immune system, we thoroughly profiled immune tissues, cells, responses to viral and bacterial challenges, and the dynamics of embryonic development in our model.

Results

We discovered the ancient expansion of FcRL genes in teleost fish. The FcRL repertoire emerged through whole-genome duplications, leading to receptors with uniquely structured domains and functional signaling motifs (ITAM/ITIM). We observed strikingly different transcriptional dynamics across tissues and immune cell types, including both systemic and mucosal lymphoid organs, in blood and leukocyte subsets. Moreover, FcRL gene expression was found to be dynamically regulated during embryonic development and throughout both innate and adaptive immune responses to pathogens.

Conclusions

The expanded FcRL family in teleosts seems to make up for their limited immunoglobulin diversity, unlocking a wider array of antibody-mediated effector functions. These groundbreaking findings offer fresh perspectives on the evolution of immune receptors in early vertebrates, challenging the conventional mammalian models and deepening our understanding of adaptive immunity in teleost fish.

1. Kyslík, Jiří, Institute of Parasitology, Biology Centre Czech Academy of Science, Czechia, Presenter
2. Rebl, Alexander, Research Institute for Farm Animal Biology (FBN), Working Group Fish Genetics, Dummerstorf, Germany, Author
3. Chan, Tze Ho Justin, Fish Health Division, University of Veterinary Medicine, Vienna, Austria, Author
4. Majstorović, Jovana, Institute of Parasitology, Biology Centre Czech Academy of Science, Czechia, Author
5. Dedić, Neira, Institute of Parasitology, Biology Centre Czech Academy of Science, Czechia, Author
6. Adamek, Mikolaj, Fish Disease Research Unit, Institute for Parasitology, University of Veterinary Medicine Hannover, Hannover, Germany, Author
7. Köllner, Bernd, Institute of Immunology, Friedrich Loeffler Institute, Federal Research Institute for Animal Health, Greifswald, Germany, Author
8. Piačková, Veronika, University of South Bohemia in České Budějovice, Faculty of Fisheries and Protection of Waters, South Bohemian Research Cen, Author
9. Korytář, Tomáš, Institute of Parasitology, Biology Centre Czech Academy of Science, Czechia, Author

Filling the gap: Starting to decrypt the genetic diversity of Aphanomyces astaci in Switzerland

Introduction
The genetic diversity of Aphanomyces astaci influences host-pathogen interactions. Genetic diversity of A. astaci in Europe is broad and five major genotype groups (A-E) have been identified. These groups have been linked to different low-susceptibility North American crayfish species, acting as carriers, and to varying disease virulence in susceptible European crayfish species.
Material and Methods
To detect these genotype groups in Switzerland, we adapted the qPCR protocol by Di Domenico et al. (2021) into a pentaplex qPCR. We analyzed DNA from fresh tissue samples (2020-2023) and archived Bouin’s or formalin-fixed, paraffin-embedded tissue samples (1991-2020).
Results and Discussion
The adapted pentaplex qPCR reliably detected genotype groups A, B, D, and E in 28 out of 46 populations. Most samples with detectable genotype groups were linked to crayfish plague outbreaks in European crayfish. Only two North American crayfish populations had detectable genotype groups, both B. No sample reacted positively with the assay for genotype group C.
The highest genotype variability was observed in the Rhine basin, peaking between 2016 and 2020. In southern Switzerland’s Ticino basin, genotype groups E and B were found, while only B was found in the Rhone basin. Genotype group A was only detected once in 2017 in the Rhine basin.
The use of archived samples allowed the detection of genotype groups back to the 1990s, revealing high genotype diversity in Switzerland and aiding understanding of crayfish plague spread in Europe.

1. JEMMI, ELIANE, UNI BERN FIWI, Presenter
2. PISANO, Simone, UNI BERN FIWI, Author
3. STEINER, Jonas, UNI BERN FIWI, Author
4. CRISTINA, Elodie, UNI BERN FIWI, Author
5. DELAFORTIE, Zoe, UNI BERN FIWI, Author
6. DELALAY, Gary, UNI BERN FIWI, Author
7. SCHMIDT-POSTHAUS, Heike, UNI BERN FIWI, Author

Dietary EPA and DHA boost viral disease resistance in Atlantic Salmon (Salmo salar)

Viral diseases represent a major health and economic challenge in global salmonid aquaculture. Diseases such as cardiomyopathy syndrome (CMS), heart and skeletal muscle inflammation (HSMI), jaundice syndrome, and pancreas disease (PD) have led to substantial losses and compromised fish welfare. Numerous studies have shown that eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) are essential for optimal salmonid growth and resilience to viral infections. This study aimed to determine whether higher dietary levels of EPA+DHA can enhance resistance to viral infections.

A 19-week feeding trial was conducted using a PD (salmonid alphavirus-3, SAV3) cohabitation challenge model at VESO Vikan, Norway. Atlantic salmon (initial weight ~25 g) were fed two isoproteic and isoenergetic diets containing two levels of EPA+DHA. After a 9-week pre-feeding (including smoltification), fish were exposed to SAV3 through cohabitation for 10 weeks in triplicate tanks. Fifteen fish per tank were biweekly sampled for post-challenge assessments of viral load in heart, plasma biomarkers including creatine kinase (CK) alanine transaminase (ALT) and aspartate transaminase (AST), and histopathological lesions in pancreas, heart, and skeletal muscle.

T-test and Mann-Whitney U analysis showed that fish fed higher levels of EPA+DHA had significantly lower SAV3 loads, reduced plasma CK, ALT, AST levels, and milder tissue lesions associated with PD. In conclusion, increasing dietary EPA+DHA enhances viral disease resistance. More details of the results will be presented and discussed at the conference.

1. GU, JINNI, BIOMAR AS, Presenter
2. SIGHOLT, TRYGVE, BIOMAR AS, Author
3. AASUM, ELISABETH, BIOMAR AS, Author

Cross-reactivity of a monoclonal antibody against CD4-2 in ginbuna crucian carp with lymphocytes from other cyprinids: common carp, grass carp and zebrafish

The CD4 molecule is a cell surface marker of helper T (Th) cells, which plays an essential role in the adaptive immune system in mammals. In teleost fish, two types of CD4 molecules have been identified: CD4-1, which is homologous to mammalian CD4, and CD4-2, which has a unique structure specialized in teleost fish. There are three CD4⁺ cell subpopulations, CD4-1 single positive (SP), CD4-2 SP and CD4 double positive (DP). Previously, we developed a monoclonal antibody (mAb), D5, against CD4-2 in ginbuna crucian carp (Carassius auratus langsdorfii), and demonstrated that CD4-1 SP and CD4-2 SP cells are two major populations in ginbuna. In this study, we investigated the cross-reactivity of D5 with other cyprinid species, including common carp, grass carp and zebrafish, and determined the composition of the CD4⁺ cell subpopulations in these species.

Thymus, trunk kidney, head kidney, spleen and peripheral blood were collected from common carp (Cyprinus carpio) and grass carp (Ctenopharyngodon idellus). Leukocyte suspensions from each tissue were isolated through centrifugation using a 1.08 g/ml Percoll solution. For zebrafish (Danio rerio), whole cell suspensions were prepared from four lymphoid tissues (thymus, trunk kidney, head kidney, and spleen) and gills. All cell suspensions were subjected to immunofluorescence staining with mAbs against ginbuna CD4-1 (6D1, provided by Prof. Teruyuki Nakanishi) and ginbuna CD4-2 (D5), followed by DAPI staining to exclude dead cells. The percentages of mAb-positive lymphocytes were analyzed by flow cytometry.

Flow cytometric analysis demonstrated that D5 reacted with lymphocytes in common carp, grass carp, and zebrafish. In all species, the percentages of CD4-1 SP and CD4-2 SP cells were higher than those of CD4 DP cells. In common carp and grass carp, CD4-1 SP cells were most abundant in the thymus (29.8%–44.1%) and least in PBL (2.4%–3.2%). CD4-2 SP cells were least abundant in the thymus (1.5%–10.6%) but present at around 25% in the other tissues. CD4 DP cells were less than 10% in all tissues. In zebrafish, CD4-1 SP, CD4-2 SP, and CD4 DP cells were present at around 6%, 17%, and 5%, respectively, in lymphoid tissues, while in the gills, they were present at around 12%, 11%, and 3%, respectively.

In conclusion, mAb D5 cross-reacts with CD4-2 in common carp, grass carp, and zebrafish. Furthermore, it was suggested that cyprinid fish, have two major CD4⁺ cell subpopulations (CD4-1 SP and CD4-2 SP) and a small population of CD4 DP cells.

1. Goto, Terumi, Tokyo University of Marine Science and Technology, Presenter
2. Lau, Lik-ming, Tokyo University of Marine Science and Technology, Author
3. Fischer, Uwe, Friedrich-loeffler-institut, Author
4. Klafack, Sandro, Friedrich-loeffler-institut, Author
5. Matsumoto, Megumi, Tokyo University of Marine Science and Technology, Author
6. Sano, Motohiko, Tokyo University of Marine Science and Technology, Author
7. Kato, Goshi, Tokyo University of Marine Science and Technology, Author

Advanced computing and camera technology-based AI-powered probe for the real-time detection of shrimp diseases

Emerging and re-emerging diseases are persistent challenges for the global shrimp industry. Sustaining shrimp health is crucial, as failure to do so can result in significant losses for farms. Traditionally, farmers assess the health status of shrimp by observing them daily, inspecting ponds to assess the presence of mortalities and clinical signs (e.g. abnormal behaviour).  This method is inherently prone to errors and delays. To address these issues, current research is leveraging Deep Learning (DL) AI models combined with advanced imaging and video camera technology.

In an ongoing project, we aim to train algorithms in detecting abnormal behavior of shrimp and presence of clinical signs recorded with high-definition underwater and submerged cameras. Camera sensors provide periodic optical monitoring of shrimp morphology and behavior, producing extensive datasets for AI training. Python is used for both data acquisition and integration with machine learning workflows due to its broad support in the field. The collected data supports various AI pipelines, including classification, object detection, segmentation, and key point detection. Live camera feeds are processed through a sequence of AI models: initial object detection for shrimp tracking, followed by identification of stress indicators or lesions, and finally, a decision model to trigger alerts for abnormal findings.

The current study evaluates the feasibility of an AI-based imaging system (Disease Detector) for real-time health monitoring in shrimp aquaculture. As a demonstrative case, white spot syndrome virus (WSSV) infection in Litopenaeus vannamei was used to test the system’s functionality. Three sequential trials were conducted to estimate the Shrimp 50% Infective Dose (SID₅₀), assess camera performance, and generate deep learning datasets for clinical signs of white spot disease (WSD). Initial findings confirm the system’s ability to capture early disease indicators, supporting its potential for timely detection and stakeholder notification. This approach enables continuous, online monitoring, offering a promising tool for precision shrimp farming.

1. UTHAMAN, SHYAM KOKKATTUNIVARTHIL, DENMARK TECHNICAL UNIVERSITY, Presenter
2. Vendramin, Niccolò, DENMARK TECHNICAL UNIVERSITY, Author
3. Stæhr, Casper, Sincere Aqua, Denmark, Author
4. Stæhr, Gustav, Sincere Aqua, Denmark, Author
5. Mellemgaard, Fridi, Sincere Aqua, Denmark, Author
6. Jensen, Britt Bang, DENMARK TECHNICAL UNIVERSITY, Author

Infection with SAV2 and SAV3 results in differences in viral loads, pancreas disease progression, and growth impact – what are the mechanisms behind the differences?

Fabian Kropp¹, Fadi Alnaji², Simen Nørstebø¹, Marco Vignuzzi², Øystein Evensen1, Turhan Markussen¹, and Aase B. Mikalsen¹

¹Faculty of Veterinary Medicine, Norwegian University of Life Sciences, Ås, Norway.

²A*STAR Infectious Diseases Labs, Immunos, Singapore

Introduction

Pancreas disease (PD), caused by salmonid alphavirus (SAV), is a major challenge for the aquaculture industry in Norway and Europe. Infected fish develop severe lesions in the pancreas as well as inflammation in the heart and skeletal muscle, leading to reduced fish welfare and increased economic losses for the producers, due to impaired growth and mortality. Disease outbreaks in Norway are caused by either the SAV3 or SAV2 genotypes of the virus. SAV3 infections are considered to have a more significant impact on fish health, welfare, and industry outcomes compared to SAV2, but the mechanisms behind this difference are not well understood.

Materials and methods

In this study, we generated SAV3 and SAV2 viruses from plasmid-based clones and infected groups of parr separately with equal viral doses of each genotype. Infected fish were monitored in a long-term study of 16 weeks. Quantitative analyses of viral RNA and concurrent histopathological assessments of the heart, muscle, and pancreas were performed weekly the first four weeks, and then every fourth week. In an attempt to decipher the underlying mechanisms producing the observed genotype-specific differences, additional analyses were included to study presence and characteristics of defective viral genomes (DVGs) over the time course and variations in host responses to SAV3 and SAV2 infection. Also, blood from infected fish sampled at different time points were also analyzed, targeting protein biomarkers associated with heart and pancreas damage and regenerative mechanisms.

Results

The in vivo experimental challenge trial demonstrates that SAV3, as expected, replicates to high levels in the fish during the first four weeks. This causes severe damage to the pancreas, seen as early as two weeks post challenge, and growth is completely halted. SAV2 replication is lower during this period and the infection causes less pancreas damage with regeneration starting as early as week four. Also, the lesions in the heart from SAV2 infected fish are less severe and almost insignificant. Targeted biomarkers in the blood show time-dependent increase with some differences observed between the genotypes. Preliminary results from analyses for the presence of DVGs, and their potential associations with viral replication and disease development, will also be presented.

Conclusions

The two SAV genotypes replicate at different rates and to different levels in the fish and induce distinct differences in PD severity and growth performance.

1. Mikalsen, Aase B., Norwegian University of Life Sciences, Author
2. Kropp, Fabian, Norwegian University of Life Sciences, Presenter
3. Alnaji, Fadi, A*STAR Infectious Diseases Labs, Author
4. Nørstebø, Simen, Norwegian University of Life Sciences, Author
5. Vignuzzi, Marco, A*STAR Infectious Diseases Labs, Author
6. Evensen, Øystein, Norwegian University of Life Sciences, Author
7. Markussen, Turhan, Norwegian University of Life Sciences, Author

Development of New Vaccines Against T. finnmarkense in Salmon in Norway

Tenacibaculosis is a disease that causes skin ulcers and mouth erosions in Atlantic salmon. This disease, caused by members of the relatively newly discovered bacterial genus Tenacibaculum, has gained increasing attention as a disease agent over the past 10–15 years. Today, it is known that Tenacibaculum spp., along with other “wound bacteria,” contribute significantly to mortality, reduced welfare, and disease reported in the Norwegian salmon industry. Tenacibaculum finnmarkense genomovar finnmarkense is the dominant etiologic agent in non-classical winter ulcers.

In this project we tested various combinations of water-based immersion vaccines (dip vaccines) and oil-based intraperitoneal vaccines (IP vaccines) against T. finnmarkense before performing a challenge study to investigate the efficacy of administered vaccines. Specific IgM response against T. Finnmarkense in serum from the vaccinated fish was investigated by ELISA.

The results demonstrated a specific IgM response against T. finnmarkense in all IP vaccinated fish. Significant differences in mortality were observed between unvaccinated control fish and vaccinated fish. Immersion vaccination in combination with IP vaccination gave no additional antibody response or additional protection compared to IP vaccinated fish alone. However, in two of three tanks in the second challenge, double immersion with IP vaccination gave additional protection compared to only one immersion with IP vaccination. In the ongoing work we will investigate immune responses in, skin, gills, thymus, nose cavity and intestine in addition to kidney and spleen to determine how immersion vaccination affect the mucosal immunity.

Our result demonstrates protection against T. finnmarkense in an experimental challenge study and our trial vaccine may become the first vaccine that can effectively assist the salmon aquaculture industry in Norway in the combat against tenacibaculosis.

1. SETERNES, TORE, UNIVERSITY OF TROMSØ, Presenter
2. Mechlaoui, Marwa, University of Tromsø, Author
3. Erikstad, Helge-Andre, Vaxxinova Norway AS, Author
4. Haganes, Tone, Vaxxinova Norway AS, Author
5. Isdal, Eivind, Vaxxinova Norway AS, Author
6. Småge, Sverre, Cermaq AS, Author
7. Dalmo, Roy A., University of Tromsø, Author