Author: marina02

Gene expression in the gills of freshwater Atlantic salmon affected by epitheliocystis

Introduction

Epitheliocystis is an intracellular bacterial infection affecting mucosal surfaces (mostly gills) of fish which can cause clinical signs and mortalities. This disease contributes to multifactorial gill diseases and can affect salmon performance. Candidatus Clavichlamydia salmonicola has been associated with epitheliocystis in freshwater Atlantic salmon. Epitheliocystis research focused on the pathogen taxonomy and epidemiology of the disease, with research on the host response during the infection limited to histology. Here, we investigated differential gene expression in gills of Atlantic salmon affected by epitheliocystis.

Methodology

Atlantic salmon smolts sampled from a recirculating aquaculture system hatchery in Ireland were diagnosed with epitheliocystis (Quezada-Rodriguez et al 2022). The severity of epitheliocystis was evaluated on the basis of histology and RNA copy number of Candidatus Clavichlamydia salmonicola. Differential gene expression analysis and gene set enrichment analysis (GSEA) were performed using reads aligned to the NCBI Ssal v3.1 release of the Atlantic salmon genome. GSEA was conducted using Gene Ontology terms from the biological process domain. Transcriptome analysis included fish with no cysts (n=6) and those with >9 cysts/gill filament (n=10) based on histological assessment. Individuals with low (n=6) and high pathogen load (n=6) as measured by species specific PCR were also compared.

Results and Conclusions

Based on principal component analysis, the fish were grouped into two clusters reflecting their strain and sampling point. To reduce the complexity of the dataset, only the fish from one origin was retained for transcriptome analysis. Sixteen differentially expressed genes (DEGs) were upregulated and 10 downregulated in the comparison of fish based on number of cysts/filament. Only 3 DEGs (all with immune function – integrin alpha-L like, Ig kappa-b4 chain C region like and chemokine XC receptor 1) were identified in low pathogen RNA/high pathogen RNA comparison. For the same comparison, GSEA revealed the enrichment of the defence response and cytokine receptor binding gene sets in the low pathogen RNA group relative to the high pathogen RNA group. Several of DEGs and gene sets in infected Atlantic salmon were similar or the same as reported for Chlamydia spp. infections, for example Chlamydia induced epithelial-mesenchyme transition (EMT), including upregulation of fibronectin, while C. trachomatis actively suppressed CXCL10. Upregulation of GTPase and tubulin suggests that similarly to Chlamydia, the epitheliocystis pathogen hijacks host’s systems to co-opt host’s cytoskeleton for the intracellular inclusion. Interferon gamma inhibited C. trachomatis invasion in vitro. This suggests that the host response against chlamydiales is conserved across vertebrates.

References

Quezada-Rodriguez, P.R., Taylor, R.S., Downes, J., Egan, F., White, S., Brenan, A., Rigby, M., Nowak, B. F., Ruane, N. M., & Wynne, J. W. (2022). Prevalence of epitheliocystis in freshwater Atlantic salmon reared in flow-through and recirculation aquaculture systems. Journal of Fish Diseases, 45, 1721–1731. https://doi.org/10.1111/jfd.13694

1. NOWAK, BARBARA, UNIVERSITY OF TASMANIA, Presenter
2. Smith, Aaron, CSIRO, Author
3. Quezada-Rodriguez, Petra, CSIRO, Author
4. Wynne, James, CSIRO, Author
5. Taylor, Richard, CSIRO, Author
6. Jantawongsri, Khattapan, Chulalongkorn University, Author
7. Ruane, Neil, Marine Institute, Author
8. White, Samantha, Marine Institute, Author
9. Downes, Jamie, Marine Institute, Author

Distribution and abundance of fish parasites in environmental water samples before a major river restoration event

After 100 years of impeding anadromous fish migration in the Klamath River (USA), the largest dam removal event in the world is now complete. The dams affected river flow and temperature, and altered the host-parasite balance in the system, with consequent detrimental impacts on salmonid populations. But how will dam removal affect waterborne parasites and their hosts, and thereby alter pathogen effects and disease in resurgent salmon populations? River reconnection means that both adult and juvenile anadromous salmonids will be exposed to novel pathogens in the upper basin, and resident upper basin fishes will be exposed to pathogens associated with the new in-migrants. Mapping parasite distributions before and after dam removal is essential to assess risks of novel and known threats to fish health, and to empower managers with information on which to base decisions. Accordingly, we collected river water for three years pre-dam removal (2021-2023), the removal year (2024), and plan to continue for another 3 years post dam removals. Each month, we sampled at 26 sites in the tributaries and mainstem of the river, in localities relevant to salmonid ecology. For each site, 3 x 1-L samples were filtered @ 0.2 µm, and total DNA extracted. We used specific qPCR assays to quantify the fish parasites Ceratonova shasta, Parvicapsula minibicornis, Ichthiophthierius multifiliis, Nanophyetus salmincola and the annelid host of two of these, Manayunkia occidentalis. We detected all five organisms in both tributary and mainstem localities, but with different temporal patterns. Waterborne stages of C. shasta were most abundant overall. C. shasta, P. minibicornis and their annelid host were most abundant in the mainstem whereas I. multifiliis and N. salmincola were more abundant in tributary sites. Successful detections show the utility of this environmental water sampling approach, and will lead to deeper analyses of the diverse parasite and host biota represented in these samples, before and after dam removals.

1. Hallett, Sascha, Oregon State University, Author
2. Maria, Müller, Oregon State University, Author
3. Atkinson, Stephen, Oregon State University, Presenter

EPIDEMIOLOGICAL MONITORING OF INFECTIOUS HEMATOPOIETIC NECROSIS VIRUS IN RAINBOW TROUT FRY AND BROODSTOCK IN A FACILITY FOR 5 YEARS

Infectious hematopoietic necrosis (IHN) poses a significant threat to salmonid aquaculture operations worldwide. In Japan, IHN remains prevalent among fry and juvenile rainbow trout in aquaculture since its invasion into the country in 70’s and the causative virus, IHNV has evolved to occur in three unique genetic lineages: JRt Nagano (JN), Shizuoka (JS), and North Kanto (JnK) from the original U genogroup. Although information on virus dynamics in the fish production cycle is needed to understand how virus genetic diversity occurs, there is little data so far. This study focuses on epidemiological monitoring of IHNV in rainbow trout Oncorhynchus mykiss at the Nagano Prefectural Fisheries Experimental Station, which received supply water contaminated with the wastewater of two aquaculture facilities upstream. IHNV was isolated in EPC cells from the ovarian fluid of broodstock, and dead and surviving fry between 2019 and 2023 were sequenced for a part of the nucleocapsid (N: 542 nt) gene and glycoprotein (G: 558 nt) genes. The phylogenetic trees were constructed from both partial gene sequences (N+G: 1,100 nt) to investigate the relationships of the virus isolates from different samples year by year. Furthermore, as an experimental epidemiology, fry artificially infected with a known virus strain were raised for approximately 18 months, after which ovarian fluids were collected during their first spawning period to examine the isolated viruses. The results revealed two dominant lineages corresponding to JN and JnK, with distinct genetic clustering of isolates across the five-year period. The isolates from dead and surviving fry, and broodstock differed genetically from each other. Moreover, the same broodstocks produced ovarian fluid containing different isolates each year. During the spawning season, an experimental epidemiology with artificial infection showed no inoculated virus in the ovarian fluid of fish, which had been experimentally infected with the virus at the fry stage, whereas the isolated virus resembled those detected in other spawning fish in the same year. These results indicate that broodfish are re-infected with the emerged viruses, which are genetically different from those in fry, at the maturation period, suggesting that virus selection occurred at the broodfish stage, and consequently, virus evolution may mostly occur in persistently infected fish at the grow-up stage.

Keywords: Infectious hematopoietic necrosis, Rainbow trout, Molecular epidemiology, Genetic diversity

1. SU THIN, CHAW, Tokyo University of Marine Science and Technology, Tokyo, Japan, Presenter
2. NONAKA, AOI, Tokyo University of Marine Science and Technology, Tokyo, Japan, Author
3. TSUBOI, GOSUKE, Tokyo University of Marine Science and Technology, Tokyo, Japan, Author
4. HIRABE, SUGURU, Tokyo University of Marine Science and Technology, Tokyo, Japan, Author
5. SHIMIZU, RYOTA, Tokyo University of Marine Science and Technology, Tokyo, Japan, Author
6. SHIROTORI, FUMIAKI, Nagano Prefectural Fisheries Experimental Station, Nagano, Japan, Author
7. TAKEUCHI, TOMOHIRO, Nagano Prefectural Fisheries Experimental Station, Nagano, Japan, Author
8. TAKEHANA, KOTA, Nagano Prefectural Fisheries Experimental Station, Nagano, Japan, Author
9. FURIHATA, MITSURU, Nagano Prefectural Fisheries Experimental Station, Nagano, Japan, Author
10. TAKIKAWA, TOMOHITO, Fuji Trout Hatchery, Shizuoka Prefectural Research Institute of Fishery and Ocean, Shizuoka, Japan, Author
11. NAKAMURA, EISUKE, Fuji Trout Hatchery, Shizuoka Prefectural Research Institute of Fishery and Ocean, Shizuoka, Japan, Author
12. MATSUMOTO, MEGUMI, Tokyo University of Marine Science and Technology, Tokyo, Japan, Author
13. KATO, GOSHI, Tokyo University of Marine Science and Technology, Tokyo, Japan, Author
14. SANO, MOTOHIKO, Tokyo University of Marine Science and Technology, Tokyo, Japan, Author

Evaluating the performance of the surveillance for pancreas disease in Norwegian salmonid farming

Introduction Scenario tree modeling is widely used in veterinary epidemiology to support estimation of surveillance system sensitivity, demonstration of disease freedom, and the design of cost-effective surveillance strategies. The current Norwegian surveillance system for pancreas disease (PD) includes both monthly sampling and testing at all marine salmonid sites and clinical investigations by veterinarians and fish health inspectors. Confirmation of PD requires detection of salmonid alphavirus by two independent laboratory methods, such as RT-PCR, serology, virus isolation, or histopathology. Between 2010 and 2020, confirmed PD cases averaged 134 annually, peaking at 176 cases in 2017. Since 2020, confirmed cases have declined sharply, reaching 48 in 2024. This trend poses challenges for detection and interpretation, as the likelihood of identifying cases in a large population decreases. It may also signal a need to reassess and optimize the current surveillance strategy. This study aimed to evaluate the performance of the current PD surveillance system, considering its components and the changing pattern of PD occurrence in Norway, including the probability of disease freedom in sites outside endemic zones.

Methodology We developed a scenario tree model (STM) for PD in marine sites using monthly surveillance data from 2019. The model incorporates test characteristics, site-level design prevalence, and site-specific relative risk based on spatial proximity and proxies for local disease pressure. These risk indicators are combined to assign a probability of infection for each site, which is subsequently used with test sensitivity to estimate site-level detection probabilities. Active surveillance (routine sampling) and passive surveillance (clinical suspicion) are included as independent components. These are aggregated to estimate system sensitivity (SSe) and update the probability of freedom (PFree) over time.

Results Preliminary results indicate that SSe remains modest across most production areas. However, PFree increases over time as evidence accumulates in the absence of detected cases. Clear variation in both SSe and PFree was observed across areas. Future work will extend the analysis to the full period since the current surveillance system was implemented (2018 to present). We also plan to evaluate alternative scenarios, include additional risk factors, assess the impact of dependence between surveillance components, and conduct cost-effectiveness analysis of selected scenarios.

Conclusions The proposed STM provides a starting point for a structured approach to assessing PD surveillance in Norway. As the model is further developed, it may help identify opportunities to improve system performance and support decision making when comparing alternative surveillance strategies.

1. Oliveira, Victor H. S., NORWEGIAN VETERINARY INSTITUTE, Presenter
2. Bang Jensen, Britt, TECHNICAL UNIVERSITY OF DENMARK, Author
3. Dean, Katharine R., NORWEGIAN VETERINARY INSTITUTE, Author
4. Walde, Cecilie S., NORWEGIAN VETERINARY INSTITUTE, Author

Enhancing Immune Protection in Threadfin Fish: A Recombinant Subunit C5a Peptidase Vaccine Combining Formalin-Killed Streptococcus iniae Cells

Streptococcus iniae is a significant pathogen in aquaculture, particularly affecting threadfin fish, leading to substantial economic losses. Effective vaccination strategies are essential to combat this pathogen while minimizing reliance on antibiotics. This study aims to develop a recombinant subunit vaccine combining recombinant C5a peptidase (C5a)and formalin-killed cells (FKC) of S. iniae to enhance immune responses in four-finger threadfin fish. The C5a gene was cloned and expressed in Escherichia coli, and the recombinant protein was purified and refolded for use in immunization. Fish were vaccinated with either C5a, FKC, or a combination of C5a and FKC. Immunological assessments included antibody titer, lysozyme activity, and gene expression analysis of immune markers. Results showed that the combination C5a and FKC group exhibited the highest antibody titer against both C5a and whole-cell S. iniae antigens, with significant increases in lysozyme activity. Gene expression analysis revealed upregulation of innate and adaptive immune markers such as CD8 and IL-10, indicating robust immune activation. Histopathological evaluations showed that vaccinated fish had significantly reduced tissue damage compared to controls. This study demonstrates that the combination C5a and FKC vaccine significantly enhances both innate and adaptive immune responses in threadfin fish, providing a promising approach to improving aquaculture vaccine efficacy against S. iniae, potentially reducing the need for antibiotics and promoting sustainable farming practices.

1. Giovanni, Andre, International Program of Ornamental Fish Technology and Aquatic Animal Health, International College, NPUST, Pingtung Taiwan, Presenter
2. Shi, Yin-Ze, Department of Agricultural Biotechnology, College of Agriculture, National Chiayi University, Chiayi, Taiwan, Author
3. Wang, Pei-Chi, Department of Veterinary Medicine, College of Veterinary Medicine, NPUST, Pingtung, Taiwan, Author
4. Tsai, Ming-An, Department of Veterinary Medicine, College of Veterinary Medicine, NPUST, Pingtung, Taiwan, Author
5. Chen, Shih-Chu, General Research Service Centre, National Pingtung University of Science and Technology, Pingtung, Taiwan, Author

A retrospective study of adverse welfare effects of handling farmed salmonids when treated for ectoparasites at sea evaluated by a Delphi panel

Norwegian legislation intended to protect wild stocks of Atlantic salmon (Salmo salar (L)) from excessive burdens of salmon lice (Lepeophtheirius salmonis) by being shedded from famed salmon at sea require systematic and frequent delousing of all fish in a net pen. Each net pen typically contains up to 200.000 individual fish. Chemotherapeutic bath and in-feed treatments have increasingly lost effect through rapid development of host resistance to the active compounds due to repeated use and a pervious all or nothing treatment strategy. Alternative mechanical, thermal and hydrodynamic methodology for delousing have been developed and have increased in its use over the past decades. Vessels, either well boats or purpose built barges with top deck utilisation of mechanical lice removal devises, water jet cleaners or thermal delousing with exposure of seawater of around 30°C for about 30 seconds are routinely used and communally termed non-medicinal delousing. These methods are controversial due to suspected negative fish welfare burden through stress, discomfort and potential for inflicting injury and pain. Our study include data collected from four fish farming companies and represents cases of handling and their outcomes. The selection criteria for the cases were predefined, hence the selection was not influenced by the data to be found. The data from actual cases were condensed into a short description of each individual scenario to be presented to a Delphi-panel consisting of a diverse group of people who are stakeholders or in other ways affiliated with salmonid farming. Results from the Delphi panel evaluation of scenarios seen as consistent and will be presented.

This study is a part of the four-year research project Welfare Severity funded by the Research Council of Norway (PNR: 326980). The project is a cooperation between the Institute of Marine Research, Nofima, the Norwegian Veterinary Institute, Wageningen University and Research and an industrial consortium. This consortium includes three industry organisations, four farming companies and two well boat holding companies. The primary goal of the project is to develop severity assessment frameworks for classifying the welfare of salmon in aquaculture.

1. Bleie, Hogne, Mallard AS, Presenter

Experience with the use of a live attenuated vaccine against SRS

Piscirickettsia salmonis causes Salmonid Rickettsial Septicemia (SRS) in farmed salmonid fish globally. In autumn 2024, the disease was diagnosed at several sea sites in Northern Norway. In Chile, SRS has been a significant problem and the main cause of antibiotic use. Comparison of genomes of P. salmonis has shown that there is considerable genetic diversity both within Chile and between Chile, Norway, and other geographies. Vaccines against SRS have been available in the Chilean market for several decades. We present our experience with the live attenuated vaccine ALPHA JECT LiVac SRS (PHARMAQ AS) in Chile, and data on genetic diversity’s impact on cross-protection. Despite genetic differences causing serological variations, vaccinated fish are protected against various P. salmonis strains in laboratory studies. This indicates that vaccination with a live attenuated P. salmonis vaccine has the potential to be a useful tool against SRS outside of Chile as well.

1. Karlsen, Marius, PHARMAQ, Presenter
2. Olsen, Rolf Hetlelid, PHARMAQ, Author
3. McAdam, Martin, PHARMAQ, Author
4. Haaland, Espen, PHARMAQ, Author
5. Santander, Claudia, PHARMAQ, Author
6. Heen, Vegard, PHARMAQ, Author
7. Bordevik, Marianne, PHARMAQ, Author
8. Buene, Glenn, PHARMAQ, Author
9. Sandlund, Liv, PHARMAQ, Author
10. Behzaad, Helen, PHARMAQ, Author

Common issues in reporting parasitic diseases in farmed fish

Introduction

Outbreaks of parasitic diseases on fish farms can affect the health of the fish and increase the costs of fish production. Reporting parasitic diseases in farmed fish is essential, it increases our knowledge and contributes to assessing the risks of disease outbreaks and developing management strategies for those diseases. However, to be helpful the reports need to be accurate. Issues can arise if the parasite is incorrectly identified (for example Neoparamoeba pemaquidensis was originally thought to cause amoebic gill disease in mariculture of Atlantic salmon but this disease is caused by Neoparamoeba perurans) or if the parasite is identified only to genus but there is a co-infection with two species (for example Cardicola forsteri and Cardicola orientalis can co-infect Southern bluefin tuna). The aim of this study was to review accuracy of reporting parasitic diseases in fish in publications from scientific journals and propose an improved approach.

Methodology

Scientific papers reporting parasitic diseases in farmed fish were identified through Web of Science. The search was done using keywords “fish parasitic disease aquaculture” limited to publications from 2000 to 2024. Issues affecting accuracy of the reports and common to at least two publications were identified.

Results and Conclusions

Based on the search, reports of parasitic diseases in farmed fish increased from 5 in 2000 to 55 in 2024. A range of common issues, present in a number of publications. For example, the parasites were misidentified or identified only to subphylum or class. In other cases, presence of DNA was assumed to be the same as the presence of the parasite. Furthermore, parasite presence was considered a disease without evaluation of disease signs. When reporting a parasitic infection, it is helpful to determine its severity. Some reports fully relied on prevalence without considering infection intensity. In this presentation solutions suggested to improve reporting of parasitic diseases in aquaculture and minimum expectation for reporting parasitic diseases will be recommended.

1. NOWAK, BARBARA, UNIVERSITY OF TASMANIA, Presenter

Serotyping of Norwegian and Scottish Yersinia ruckeri strains – antibody-based methods for O-serotyping

Yersinia ruckeri is a Gram negative bacterium that causes the disease Enteric RedMouth (ERM) disease in trout and yersiniosis in salmon. The classification systems for this pathogen are many and confusing and not very consistent. Terminology is frequently misunderstood, and O-serotypes sometimes denoted without the proper tools (eg specific antibodies) necessary to determine O-antigen reactivity correctly. LPS was previously demonstrated to be the protective antigen of Y. ruckeri (Welch and LaPatra, 2016) and proper O-serotyping is therefore important for determining vaccine suitability.

We have worked with O-serotyping different Y. ruckeri field strains based on monoclonal as well as polyclonal antibodies. The antibodies used have been developed in-house. Relevant reference strains were used to confirm O-serotype of unknown samples. Serotyping studies have been performed using slide-agglutination tests, ELISA and Western blot. Results obtained when using polyclonal antibodies generated in rabbit show that Y. ruckeri strains with different O-serotype have many surface structures in common that are causing high degree of cross-reactivity. However, when removing all the cross-binding proteins using enzymes, we are left with unique carbohydrate structures of O-antigen resemblance, seen as a ladder-like pattern in Western blot. Thus, it is possible to have correct annotation of O-serotype even when using polyclonal antibodies with high degree of cross-reactivity.

Results and methods used for serotyping will be discussed. Our studies show that salmon infections with Y. ruckeri are dominated by the O1b serotype in Norway and Scotland. O1a serotype has never been detected in Norway and infrequently in salmon in Scotland. Only a few samples were positive for O2 reactivity. We have not seen evidence of any other serotypes in salmon farming so far in Norway nor Scotland, including O5 and O8. Widespread O1b injection vaccination of salmon has been conducted in Norway since 2020 to obtain protection post sea transfer, and to date no clinical disease on injection vaccinated fish has been detected, and the injectable vaccine has so far been able to completely control yersiniosis outbreaks in Norwegian salmon farming post sea transfer.

1. GAUSDAL TINGBØ, MONICA, PHARMAQ PART OF ZOETIS, Presenter
2. Sandtrø, Ane, Pharmaq part of Zoetis, Author
3. Buene, Glenn, Pharmaq part of Zoetis, Author
4. Hardersen, Hege, Pharmaq part of Zoetis, Author
5. Brudeseth, Bjørn, Pharmaq part of Zoetis, Author
6. Shahin, Khalid, Pharmaq part of Zoetis, Author
7. Matthews, Christopher, Pharmaq part of Zoetis, Author

Gill Microbiome as a Sentinel for Wild Fish Health and Pathogen Surveillance Across Marine Ecosystems

Recent advances in next-generation sequencing (NGS) have enabled deeper insights into the microbial communities inhabiting fish gills—a key mucosal interface directly interacting with the aquatic environment. Here, we synthesize findings from two parallel surveys of wild fish species conducted in the Eastern Mediterranean and South China Sea, aiming to characterize gill microbiota and assess their utility as indicators for ecosystem health and pathogen prevalence.

In the Mediterranean study, 89 asymptomatic specimens representing three fish species were sampled during a research trawling expedition. In contrast, the South China Sea study analyzed 36 fish purchased from local markets. In both cases, gill tissue underwent 16S rRNA amplicon sequencing and bioinformatic analysis. Results revealed that gill microbiota composition exhibited strong host species specificity and reflected environmental gradients, including distinctions between demersal and pelagic-amphidromous species.

1. MORICK, DANNY, The University of Haifa, Presenter
2. ITAI, PELEG, The University of Haifa, Author
3. LAU, Stanley, The Hong Kong University of Science and Technology, Author
4. Davidovich, NADAV, The University of Haifa, Author