Author: marina02

Yersinia ruckeri IN CANADA: ISOLATE CHARACTERIZATION, VACCINE SAFETY AND CHALLENGE STUDIES

Yersinosis or Enteric Red Mouth (ERM) disease in fish caused by Yersinia ruckeri (Y. ruckeri), is a common bacterial septicemia that has been found in most areas of the world where salmonids are cultured in cold or temperate waters.

In recent years Y. ruckeri detections and yersiniosis outbreaks have been an increasing problem in larger fish in the sea, particularly in Norway. This disease pattern has also been reported as an increasing concern in Canada.

In this study we have characterized several Canadian Y. ruckeri field isolates from multiple regions in both Western and Eastern Canada using Western blotting. Secondly, in the laboratory and field we have assessed the safety profile when PHARMAQ’s water-based yersiniosis vaccine (ALPHA ERM Salar) is co-injected with oil-based commercial vaccines.  Furthermore, we have analyzed the cross-protection of ALPHA ERM Salar against various Y. ruckeri serotypes in laboratory challenges.

Our results show that Canadian Y. ruckeri field isolates are more heterogenous than those found in Norway.  The laboratory and field safety studies show that ALPHA ERM Salar is safe to use in combination with other commercial oil-based vaccines.  Lastly, PHARMAQ’s ALPHA ERM Salar vaccine demonstrated excellent cross-protection against 01a serotype (biotype 1 and 2).

1. Sandtrø, Ane, ZOETIS CANADA, Presenter
2. NESS, MICHAEL, ZOETIS CANADA, Author
3. Gausdal Tingbø, Monica, ZOETIS CANADA, Author
4. Buene, Glenn, ZOETIS CANADA, Author
5. Peach, Randy, ZOETIS CANADA, Author
6. SEKKELSTEN KINDT, MATS MARTIN, ZOETIS CANADA, Author

How to define epidemiological areas for surveillance of notifiable mollusc diseases – an example from Denmark

Introduction

The protozoan parasites Bonamia ostreae, Bonamia exitiosa and Marteilia refringens are causes of notifiable diseases in the EU. They infect bivalve molluscs and can cause considerable mortality. In Denmark, B. ostreae is present in the Limfjorden, in the Northern part of Jutland, but to date it has not been found anywhere else in the country. Neither of the other two parasites have been found in Denmark. A surveillance program for the three diseases was implemented in Limfjorden in 2000, as this is the major mollusc production area. After the finding of B. ostreae in the central part of Limfjorden in 2014, the surveillance program was restricted to only cover Nissum Bredning, the western part of Limfjorden. The surveillance was finally suspended in 2018, once B. ostreae was found in Nissum Bredning. Overall, the Danish waters (Limfjorden as well as other waters) cannot be considered one epidemiological unit for the three mollusc diseases, but mollusc treatment facilities receive mussels and other molluscs from several areas in the country. Further, restoration projects would like to move molluscs between areas. There is therefore a need to define epidemiological areas within the Danish waters.

Methodology

We have used available resources on the occurrence of suitable habitats for susceptible species in Denmark, based on maps of water depth. Information on hydrological and geographical properties of individual areas has been obtained from Sanitary Survey reports made for assessment of microbiological contamination of bivalve molluscs for human consumption. In addition, knowledge on prevalence of B. ostreae in Denmark and of the three diseases in other areas have been used.

Results

A map with proposed epidemiological areas for surveillance of the notifiable mollusc diseases will be presented.

Conclusions

Use of already available and easily accessible data and maps can be used for designing surveillance programs, when advanced epidemiological modelling is not an option -either due to capacity constraints or lack of data for input.

1. Bang Jensen, Britt, DTU AQUA, Presenter
2. Saurel, Camille, DTU AQUA, Author
3. Madsen, Lone, DTU AQUA, Author

Field safety and efficacy of a new Moritella viscosa vaccine for Atlantic salmon tested in large scale GCP field studies in Norway

Winter ulcers caused by the bacterium Moritella viscosa (M. viscosa) represent a severe animal welfare challenge with significant economic consequences for salmon farming in the northern hemisphere, especially when sea temperatures are low in the winter. The disease causes superficial skin lesions that can develop into deep, chronic ulcers and finally terminal septicemia. In addition to the obvious welfare challenges related to the lesions, the ulcers will also cause significant downgrade at slaughter and the need for further processing of the fish. Effective measures to prevent and treat winter ulcer disease are important to ensure the best animal welfare and economic sustainability.

Most farmed salmon in Norway is vaccinated with multivalent core vaccines that contain a classic M. viscosa component. During the last 10 years there have been increasing numbers of reports of winter ulcer disease caused by new emerging strains that are of a different serotype than the old types. PHARMAQ part of Zoetis has developed a new vaccine based on one of the new emerging strains, referred to as a variant strain. The new winter ulcer disease vaccine has gone through a thorough pre-clinical (laboratory studies) programme as well as a large-scale GCP clinical field study involving 11 commercial sea sites and 4.8 mill fish were vaccinated with the Moritella test vaccine. The results demonstrate good safety and efficacy profiles of the new vaccine, including significant reduction in mortality, ulceration and downgrade at slaughter. Detailed safety and efficacy results from the clinical field study will be presented and discussed.

The pre-clinical studies and the use of research animals were approved by the Norwegian Food Authorities and studies were performed in accordance with procedures approved by Zoetis Animal Ethics Committee.

1. GAUSDAL TINGBØ, MONICA, PHARMAQ PART OF ZOETIS, Presenter
2. Sandtrø, Ane, Pharmaq part of Zoetis, Author
3. Sekkenes, Hege, Pharmaq part of Zoetis, Author
4. Ellingsen, Bjørn, Pharmaq part of Zoetis, Author
5. Alexandersen, Svein, Pharmaq part of Zoetis, Author
6. Tønnessen, Øyvind, Pharmaq part of Zoetis, Author
7. Rajan, Binoy, Pharmaq part of Zoetis, Author
8. Jørgensen, Lars, Pharmaq part of Zoetis, Author
9. Ingrid, Hagerup, Pharmaq part of Zoetis, Author
10. Leknes, Lisa Marie, Pharmaq part of Zoetis, Author
11. Furevik, Anette, Pharmaq part of Zoetis, Author

In silico analyses of the Phocoenobacter atlanticus subspecies atlanticus subsp. nov. genome- insight into virulence factors

Introduction: Outbreaks of pasteurellosis in Norwegian Atlantic salmon have become more frequent since 2018, threatening fish welfare and diminishing sustainable aquaculture practices. Currently autogenic vaccines are used in some areas, but genomic characterisation of bacterial surface proteins, virulence factors, and immunogens will be essential for the development of efficient preventative solutions, and to better understand disease progression.

Methodology: The genome of a clinical Phocoenobacter atlanticus subsp. atlanticus subsp. nov. isolate was sequenced and annotated. Bioinformatic tools and databases were utilized to identify bacterial proteins that could be potential immunogens, hence vaccine targets. The criteria included outer membrane and/or extracellular localisation, adhesin potential, the presence of transmembrane helices and the presence of B cell epitopes. The highest ranking adhesins were further analysed to predict epitope docking of the identified epitopes with the Atlantic salmon MHC class II complex.

Results: In silico analysis revealed 58 outer membrane and extracellular bacterial proteins, of which 16 are adhesins. These are widely recognised to be virulence factors and suitable vaccine targets. A number of B cell epitopes were identified for 12 of these adhesins. Protein-peptide docking with Atlantic salmon MHC II resulted in predictions of binding affinities and dissociation constants that fall within acceptable regions, further indicating their potential immunogenic properties.

Conclusions: In future work, selected proteins from these analyses will be investigated through in vitro experiments and serological techniques in order to confirm the in silico results. Based on these functional analyses, the strongest target candidates will be prioritized in vaccine development efforts to prevent future pasteurellosis outbreaks.

1. Ellul, Rebecca, Department of Biological Sciences, University of Bergen, Norway, Presenter
2. Tselepidaki, Charoula, Institute of Marine Biology, Biotechnology and Aquaculture (IMBBC), Hellenic Centre for Marine Research, Heraklion, Greece, Author
3. Dahle, Håkon, Department of Biological Sciences, University of Bergen, Norway, Author
4. Gulla, Snorre, Norwegian Veterinary Institute, Ullevålsveien 68, P.O. Box 8146 Dep, N-0033 Oslo, Norway, Author
5. Frantzen, Cyril, ACD Pharma, Magnus Falsens vei 18, 1433 Ås, Norway, Author
6. Rønneseth, Anita, Department of Biological Sciences, University of Bergen, Norway, Author

Temporal changes in the microbiome of Salmon lice (Lepeophtheirus salmonis): Detection of potential salmonid pathogens

Ectoparasites that penetrate the epidermis of their hosts have the potential to act as biological or mechanical vectors for host pathogens and, in some cases, even serve as reservoirs. Crustacean ectoparasites of fish have been identified as potential vectors of various pathogens—an especially important role for obligate pathogens (e.g., Aeromonas salmonicida) that exhibit limited survival in seawater. Ectoparasitic salmon lice (Lepeophtheirus salmonis), which affect Atlantic salmon, can cause dermal tissue damage and, in severe cases, facilitate secondary infections, leading to substantial economic losses in salmon production. While the physical impact of sea lice is well documented, their role in pathogen transmission remains largely unclear.

We examined the microbiome of lice collected over a four-month period as part of the National Sea Lice Monitoring Programme at a salmon farm in Ireland. Microbiome analysis was conducted using two sequencing technologies: Illumina MiSeq and Oxford Nanopore Technologies (ONT) MinION. Cross-validation between platforms ensured accuracy and reliability of taxonomic assignments. In addition, the suitability of ONT for rapid pathogen screening was assessed.

The microbiome analysis revealed the presence of several genera containing known fish pathogens in L. salmonis, including Tenacibaculum, Francisella, and Vibrio. Additionally, fluctuations in the abundance of potential pathogenic bacterial genera were observed over the four-month sampling period, indicating dynamic shifts within the lice-associated microbial community.

Our work provides a comprehensive microbiome study of L. salmonis from a commercial salmon farm in the North Atlantic, highlighting its potential role in pathogen transmission. Detecting pathogens within the louse microbiome is a critical step toward determining whether lice can function as vectors or reservoirs, with important implications for disease monitoring, management, and prevention in salmon aquaculture.

1. Klimesova, Bela, Atlantic Technological University, Presenter
2. O’Dwyer, Katie, Atlantic Technological University, Author
3. Ruane, Neil, Marine Institute, Author
4. Lyashevska, Olga, Atlantic Technological University, Author
5. Rodger, Hamish, PatoGen FHS Ltd., Author
6. Talbot, Anita, Atlantic Technological University, Author

Antiviral protection in the Pacific oyster Crassostrea (Magallana) gigas against OsHV-1 infection using UV-inactivated virus

The increase of the frequency and severity of marine diseases affecting farmed marine mollusks are currently threatening the sustainability of this aquaculture sector, with few available prophylactic or therapeutic solutions. Recent advances have shown that the innate immune system of invertebrates can develop memory mechanisms allowing for efficient protection against pathogens. These properties have been called innate immune memory, immune priming or trained immunity. Previous results demonstrated the possibility to elicit antiviral immune priming to protect Pacific oysters against the ostreid herpes virus 1 (OsHV-1), currently plaguing M. gigas production worldwide. Here, we demonstrate that UV-inactivated OsHV-1 is also a potent elicitor of immune priming. Previous exposure to the inactivated virus was able to efficiently protect oysters against OsHV-1, significantly increasing oyster survival. We demonstrate that this exposure blocked viral replication and was able to induce antiviral gene expression potentially involved in controlling the infection. Finally, we show that this phenomenon can persist for at least 3 months, suggesting the induction of innate immune memory mechanisms. This study unravels new ways to train the Pacific oyster immune system that could represent an opportunity to develop new prophylactic strategies to improve health and to sustain the development of marine mollusk aquaculture.

1. MORGA, BENJAMIN, IFREMER, Presenter
2. MEGE, MICKAEL, IFREMER, Author
3. FAURY, NICOLE, IFREMER, Author
4. DEGREMONT, LIONEL, IFREMER, Author
5. PETTON, BRUNO, IFREMER, Author
6. PEPIN, JEAN-FRANCOIS, IFREMER, Author
7. RENAULT, TRISTAN, IFREMER, Author
8. MONTAGNANI, CAROLINE, IFREMER, Author

Genomic characterization of Salmon Gill Pox virus in Iceland

The Salmon Gill Poxvirus (SGPV) is known to cause an acute respiratory disease, sometimes leading to high mortality in juvenile farmed Atlantic salmon (Salmo salar L.). In 2015, the whole genome of SGPV was characterized and published, providing insight into the genetic structure of the virus.

Since 2018, samples from farmed Atlantic salmon have been screened for SGPV at Keldur. However, knowledge about the genetic composition of SGPV in Iceland and its presence in wild salmonids has remained limited.

The aim of the project was to sequence the highly conserved major capsule protein (MCP) and three genetically variable regions, V15, V16 and V27, from SGPV-positive samples collected 2018-2024 from farmed Atlantic salmon. Furthermore, to screen wild salmonids caught in the autumn of 2024 for SGPV by qPCR and sequence any positive samples obtained.

SGPV was detected in samples from both wild and farmed Atlantic salmon. Samples from other salmonids were negative. Sequencing and comparison to published SGPV showed highest similarity to Scottish and Faroese SGPV variants. Phylogenetic analysis revealed that there are at least two SGPV variants in Iceland.

This project provided a foundation for further research of the genetic diversity of SGPV in wild and farmed Atlantic salmon in Iceland.

1. SIGURÐARDÓTTIR, HEIÐA, INSTITUTE FOR EXPERIMENTAL PATHOLOGY AT KELDUR, Presenter
2. Costa, Claire, Clermint Auvergne University, Author
3. Casás Casal, Samuel, INSTITUTE FOR EXPERIMENTAL PATHOLOGY AT KELDUR, Author
4. Bjornsdottir, Þórunn Sóley, INSTITUTE FOR EXPERIMENTAL PATHOLOGY AT KELDUR, Author
5. Oddsdottir, Gudrun DÍa, University of Iceland, Author
6. Bragason, Birkir Þór, INSTITUTE FOR EXPERIMENTAL PATHOLOGY AT KELDUR, Author
7. Kristmundsson, Árni, INSTITUTE FOR EXPERIMENTAL PATHOLOGY AT KELDUR, Author

Evaluating the Immune Response of Atlantic Salmon, Salmo salar, Coinfected with Viral (Piscine orthoreovirus and Piscine myocarditis virus) and Parasitic Pathogens (Sea Lice – Lepeoptheirus salmonis)

Atlantic salmon aquaculture faces significant challenges due to the spread of infectious diseases and seasonal fluctuations in parasite abundance. Two viruses of concern—Piscine orthoreovirus (PRV-1) and Piscine myocarditis virus (PMCV) – cause severe cardiac diseases associated with mortality and reduced welfare. Sea lice (Lepeoptheirus salmonis), the most common ectoparasite to infect salmon, cause extensive skin damage and increase susceptibility to secondary infections. Despite the high prevalence of both viral pathogens and sea lice, little is known about the influence of coinfection on the teleost immune response and disease pathogenesis.

This study aims to characterize the immune response at molecular and protein levels, and monitor trends in viral kinetics, cardiac lesions, and sea lice burden during coinfection in Atlantic salmon. Before coinfection experiments, PRV-1 and PMCV challenge models are being developed in vivo. A Canadian PRV-1 strain and Norwegian PMCV isolate were intraperitoneally (IP) injected into pre-smolt Atlantic salmon and viral load (RNA) was quantified through RT-qPCR. PRV-1 load peaked in red blood cells at 5wpc with 3.1×108 copies/μg RNA, while PMCV peaked in heart tissue at 7wpc with 6.9×106 copies/μg RNA. Primers targeting genes of key pro-inflammatory cytokines, anti-viral molecules, and T cell responses are currently being optimized using tissue from these preliminary experiments. Gene expression analyses will complement existing ELISAs to detect salmonid specific IL-1β, IFN-I, IFN-γ, IL-2, IgT and IgM.

Our results show high viral replication in vivo and justify advancing to coinfection experiments to investigate how the host immune response is modulated during viral and parasitic coinfection scenarios, and influence the pathogenesis of CMS, HSMI, and sea lice infestation.

1. SABBADIN ZANUZZO, FABIO, ONDA, Presenter
2. Sonier, Donnie, ONDA & University of Waterloo, Author
3. Nixon, Brian, University of Waterloo, Author

Whole Genome Sequencing Reveals Genomic Characteristics of the Croatian Tenacibaculum maritimum Strains

Introduction: The expansion of aquaculture, compounded by climate change, has led to a rise in infectious disease outbreaks, significantly impacting fish health and surrounding ecosystems. Among the key pathogens is Tenacibaculum maritimum, a Gram-negative marine bacterium causing tenacibaculosis in a wide range of fish species globally. Despite its significance, the bacterium’s virulence mechanisms remain poorly understood, and current disease management relies heavily on antibiotics, raising concerns about antimicrobial resistance. This study investigates a T. maritimum strains isolated from European seabass (Dicentrarchus labrax) in the Eastern Adriatic Sea, aiming to uncover genetic factors related to virulence, resistance, and adaptation through whole genome analysis.

Methodology: In this study, bacterial strains were isolated from diseased European seabass and cultured on FMM agar to obtain pure colonies. The species was identified through 16S rRNA gene amplification, sequencing, and BLAST analysis. Whole genome sequencing was conducted using the Illumina HiSeq platform, and reads were quality-checked, trimmed, and assembled with SPAdes. Phylogenetic relationships were analyzed using the gyrB gene with Neighbor-Joining trees constructed in Geneious Prime.

Results: We conducted whole genome sequencing and bioinformatic analyses of T. maritimum strains isolated from clinically affected farmed European seabass. Our research identified several genes associated with the bacterium’s adaptive immune system, virulence, and antibiotic resistance. Specifically, we discovered genes involved in adhesion, biofilm formation, toxic activity, nutrient acquisition, and resistance to host defenses. The analysis of antimicrobial resistance genes revealed complex resistance mechanisms, including diverse efflux pump systems and regulatory pathways that allow T. maritimum to evade glycopeptide antibiotics and other antimicrobial agents, complicating effective treatment in aquaculture settings.

Conclusion: The insights gained from our study, particularly on the bacterial adaptive immune system, virulence-associated genes, and antibiotic resistance genes, along with the phylogenetic analysis of available strains, could guide the development of targeted strategies for managing T. maritimum infections. This research lays the groundwork for future efforts to control this pathogen and mitigate its impact on marine aquaculture.

1. GRBIN, DOROTEA, CROATIAN VETERINARY INSTITUTE, Presenter
2. ZUPIČIĆ, IVANA GIOVANNA, CROATIAN VETERINARY INSTITUTE, Author
3. ALFIER, MATEA, CROATIAN VETERINARY INSTITUTE, Author
4. ORAIĆ, DRAŽEN, CROATIAN VETERINARY INSTITUTE, Author
5. KRIŽANOVIĆ, KREŠIMIR, FACULTY OF ELECTRICAL ENGINEERING AND COMPUTING, UNIVERSITY OF ZAGREB, Author
6. ZRNČIĆ, SNJEŽANA, CROATIAN VETERINARY INSTITUTE, Author

Shedding light on the frontal filament structure and composition of Lepeophtheirus salmonis salmonis

Introduction:

L. salmonis is a parasitic copepod prevalent in the northern hemisphere that infects salmonids.  Its life cycle begins with two nauplii stages, followed by the copepodid stage, two chalimus stages as well as two preadult stages before the final molt into the adult stage. During the chalimus stages, the salmon louse firmly attaches itself to its host with a frontal filament, which allows it to stay attached to the host’s skin. In addition, a filament is formed between each stage after chalimus to keep the louse on the host during molting. Previous studies using high-resolution microscopy images have described the development and structure of the filament, showing that it is likely composed of two protein substances secreted by different groups of cells in the cephalothorax. As we lack knowledge about the filament’s composition and the genes involved in its formation, this study aims to characterize genes involved in the frontal filament formation.

Methods:

We performed RNA sequencing on a time series of L. salmonis salmonis covering the copepodid and chalimus I stages, and proteomics applied on frontal filament-enriched samples to identify filament-related candidate genes. We further used in situ hybridization (ISH) to localize candidate gene expression, and the function was evaluated by RNA interference (RNAi) experiments.

Results:

By combining RNA sequencing- and proteomic data, we identified several candidate genes potentially involved in the formation of the frontal filament. Seven of these genes were confirmed to be expressed in filament-substance secreting cells within the salmon louse cephalothorax. The ISH results suggest that frontal filament formation might be more complex than initially postulated. We also conducted RNAi to confirm the involvement of candidate genes in frontal filament formation, where three genes seem to be involved in forming the frontal filament basal plate. Further experiments are needed to determine the precise role of each of these genes.

Conclusions:

Here for the first time, genes involved in the formation of the frontal filament of L. salmonis could be identified. These seem to play a role in the formation of the basal plate, the substance connecting the filament to the surface of the fish and are thus crucial for the attachment of the louse. These findings could be useful for vaccine development against the salmon louse as the filament proteins could represent interesting antigens.  However, more research is needed to determine the exact role of each gene in the basal plate formation.

1. Comorge, Virginie, University of Bergen, Presenter
2. Eichner, Christiane, University of Bergen, Author
3. Hamre, Lars Are, University of Bergen, Author
4. Øvergård, Aina-Cathrine, University of Bergen, Author