Author: marina02

Water quality, off-flavors, and fish health in Recirculating Aquaculture Systems (RAS): Advances in high-throughput chip technology for non-invasive monitoring

Recirculating aquaculture systems (RAS) are an environmentally sustainable approach to fish production, enabling the recirculation of up to 100% of water. These closed systems ensure a controlled environment while regulating nutrient discharge. While RAS can be highly efficient, they also pose significant challenges related to (1) water quality, (2) off-flavors, and (3) disease management.

The Innovative Training Network (ITN) project RASOPTA – Safeguarding future production of fish in aquaculture systems with water recirculation (2021–2025), funded by the EU Horizon 2020 Marie Skłodowska-Curie Actions, addresses these critical issues. The project involves 12 PhD researchers across Europe who are investigating solutions to optimize RAS performance.

A key component of RASOPTA is the development of a high-throughput Fluidigm platform designed to assess water quality, off-flavors, and pathogenic organisms using environmental DNA (eDNA) and RNA (eRNA) from water samples. We present the latest advancements in the chip technology and discuss its potential in detecting co-infections, and also evaluating its potential for comprehensive pathogen surveillance in recirculating aquaculture systems.

1. JØRGENSEN, LOUISE VON GERSDORFF, UNIVERSITY OF COPENHAGEN, Presenter
2. Li, Hanxi, Technical University of Denmark, Author
3. Heegaard, Peter, Technical University of Denmark, Author
4. Larsen, Helene, Technical University of Denmark, Author
5. Jørgensen, Niels O.G., University of Copenhagen, Author

Comparative evaluation of qPCR performed by local fish health services using small-scale equipment and kits

During the Covid-19 epidemic, novel, small scale equipment and kits for performing quantitative PCR tests were developed and used in local testing, especially in Asian countries. In collaboration with an Asian kit supplier and 3 fish health services, we have evaluated the use of such tools to monitor infections of seawater reared Atlantic salmon. Samples obtained during site visits were split with one part being qPCR tested in the laboratory of the local FHS, while parallel portions of the same tissue were submitted to Aquamedics office in Oslo, and also to one of the few central laboratories that offer qPCR tests nationally.

Validating the equipment and kits for 6 viral infections of salmonids (ISAV, IPNV, SAV-3, SAV-2, PRV and was successful, as were the evaluation for 2 bacterial infections (BKD, SRS) and for 4 gill infections of salmon (cand. Branchiomonas cysticola, Desmozoon lepeophtheiri, Neoparamoeba perurans and Salmon Gill Pox virus). Comparisons will be presented and discussed, demonstrating the agreement both qualitatively and quantitatively.

An example of using the small-scale equipment and kits on samples from clinically affected fishes will be given, supporting the usefulness for differential diagnosis purposes.

After returning from a farm visit to the local office, running up to 32 (for one agent) or 16 (for two agents) samples can be performed within 3 hours, meaning that the PCR outcomes can be ready the same evening (if urgent) or, routinely, before noon the next day. This is of particular interest for field veterinary service provision in aquaculture regions where long-distance sample submission routinely causes two or more days of delay before receiving the test results.

1. Midtlyng, Paul J., Aquamedic AS, Presenter
2. Spanos, Georgios, Aquamedic AS, Author
3. Garnes-Gutvik, Sofie N., Emilsen Fisk AS, Author
4. Kunickiene, Erika, Aqua Kompetanse AS, Author
5. Ottesen, Kristin, HaVet AS, Author

Vertebral Deformities in Salmo salar and Oncorhynchus kisutch in Chile during 2023 and 2024. Diagnosis and prognosis based on improved radiological procedures.

Vertebral Deformities in Salmo salar and Oncorhynchus kisutch in Chile during 2023 and 2024. Diagnosis and prognosis based on improved radiological procedures.

Carlos Sandoval¹, Javiera Villarroel¹, Juan Pablo Prada¹, P. Eckhard Witten²

1. Veterinary Histopathology Center (VeHiCe SpA), Libertad 590, Puerto Montt, Chile.

2. Ghent University, Biology Department, Ledeganckstraat 35, B-9000 Ghent, Belgium.

ABSTRACT

Skeletal deformities have been recognized as a substantial welfare and economic challenges for the salmon farming industry. This study assesses prevalence and characters of vertebral deformities in Salmo salar and Oncorhynchus kisutch farmed in Chile in 2023 and 2024, following the classification of Witten et al., (2009), that distinguishes 20 types of vertebral column deformities. Prevalence of deformity types was obtained through the analysis of 1972 samples submitted to the Veterinary Histopathology Center (VeHiCe SpA). Specimens were categorized by year, species, and stage of development. Standard and high resolution radiology (Faxitron imaging) was used to identify types and locations of vertebral deformities. Results revealed an overall deformity prevalence of 74.5% in S. salar and 77.8% in O. kisutch, with abdominal vertebral bodies being most frequently affected in both species. The majority of alterations corresponds to fusion and compression of vertebral bodies, characterized by reduced or absent intervertebral spaces (Type 1) and by homogeneous compression (Type 2). Vertebral body fusions can aggravate into large fusion centra with impact on the muscle quality. However, not all malformations progress. Some types of malformations can be contained or even disappears (Drábikova et al., 2022). High resolution x-ray monitoring is an important tool for diagnosing, for monitoring and for the implementation of management strategies to reduce the incidence of skeletal malformations, particular those that aggravate and eventually severely impact animal welfare and product qualify.

Keywords: Spinal deformities, Radiology, Vertebral column, Salmo salar, Oncorhynchus kisutch.

References:

Drábiková et al. (2022) Aquaculture 559, 738430. doi.org/10.1016/j.aquaculture.2022.738430

Witten et al. (2009) Aquaculture, 295, 6-14. doi.org/10.1016/j.aquaculture.2009.06.037

1. VILLARROEL, JAVIERA, VEHICE, Author
2. SANDOVAL, CARLOS, VEHICE, Presenter

SNP-Based High-Resolution Melting Analysis for Precise Differentiation of Edwardsiella anguillarum and Edwardsiella piscicida in Aquaculture

This study aimed to establish a molecular method for distinguishing between two closely related Edwardsiella species, Edwardsiella anguillarum and Edwardsiella piscicida, both of which are responsible for Edwardsiellosis in fish aquaculture. To investigate their genetic differentiation, whole-genome single nucleotide polymorphism (SNP) analysis was performed, comparing E. piscicida, E. anguillarum, and Edwardsiella ictaluri. The resulting SNP distance matrices and phylogenetic analyses revealed that isolates of each species formed distinct clusters, confirming their genetic separation based on SNP variations. Further annotation of SNPs and comparative genetic analyses highlighted several genes—artI, dnaK, hlyB, ubiB, hutW, ydgD, MCP, and fliC—that exhibited significant sequence divergence between E. anguillarum and E. piscicida. Specific primers targeting SNPs in these genes were designed and evaluated using real-time PCR and High-Resolution Melting (HRM) analysis. HRM profiling revealed distinct melting temperature (Tm) peaks for E. anguillarum and E. piscicida, allowing for precise species differentiation. To further validate this approach, HRM analysis was applied to 34 bacterial isolates previously classified as Edwardsiella tarda based on phenotypic characteristics. The results reclassified 32 isolates as E. anguillarum and 2 as E. piscicida, demonstrating the effectiveness of SNP-based HRM analysis in species identification. Among the candidate genes, hlyB was identified as the most reliable marker for differentiating E. anguillarum from E. piscicida. This study underscores the utility of HRM analysis as a rapid, cost-effective molecular tool for accurately distinguishing between these closely related Edwardsiella species. Moreover, it provides valuable insights into the genetic diversity within the genus, contributing to improved diagnostics and epidemiological studies in aquaculture.

1. WANG, PEI-CHI, NATIONAL PINGTUNG UNIVERSITY OF SCIENCE AND TECHNOLOGY, Presenter
2. Rahmawaty, Atiek, NATIONAL PINGTUNG UNIVERSITY OF SCIENCE AND TECHNOLOGY, Author
3. Cheng, Li-Wu, NATIONAL PINGTUNG UNIVERSITY OF SCIENCE AND TECHNOLOGY, Author
4. Chen, Shih-Chu, NATIONAL PINGTUNG UNIVERSITY OF SCIENCE AND TECHNOLOGY, Author

Coinfection process with tenacibaculosis caused by Tenacibaculum spp.: first report on Atlantic salmon (Salmo salar L.) microbiome from Chilean salmon farms

Tenacibaculosis is an ulcerative, external disease caused by various bacterial species of the Tenacibaculum genus, collectively referred to as the “Tenacibaculum Complex.” It represents the second leading cause of mortality in Atlantic salmon (Salmo salar) in Chile, accounting for 32.9% of deaths in 2023. In the Magallanes Region (Chilean Patagonia), Bacterial Kidney Disease, caused by Renibacterium salmoninarum, is endemic; however, in recent years, coinfection outbreaks with tenacibaculosis have been reported. To investigate the bacterial structure and composition of these coinfections, a metagenomic study was conducted using 16S rRNA gene sequencing. MinION nanopore technology analyzed muscle and kidney samples of 10 diseased Atlantic salmon from fish farms in southern Chile during tenacibaculosis outbreaks. The muscle and kidney microbiomes of S. salar formed two distinctive clusters, as indicated by beta diversity measurements, with greater alpha diversity for muscle microbiomes. Fish microbiomes were mostly composed of 16 bacterial species (relative abundance > 0.04), such as Acinetobacter johnsonii, Arcobacter lekithochrous, Arcobacter sp. LPB0137, Tenacibaculum dicentrarchi, M. viscosa, and Pyrolobus fumarii, among others. T. dicentrarchi was dominant in muscle samples compared to relative abundance in kidney samples. None of the samples showed the presence of R. salmoninarum. Interestingly, M. viscosa was also detected accompanying the infectious microbiome driven by T. dicentrarchi, at least regarding relative abundance. Future studies employing a culture-based isolation strategy are necessary to emulate co-infectious sceneries in in vivo experiments between T. dicentrarchi and M. viscosa to elucidate potential synergistic relationships and to determine subsequent impacts to the Chilean salmon industry.

Acknowledgements: FONDECYT Regular N° 1230068 and FONDAP No. 1523A0007.

1. AVENDAÑO-HERRERA, RUBEN, UNIVERSIDAD ANDRÉS BELLO, Presenter
2. Levipan, Héctor, Universidad de Playa Ancha, Author
3. Araya-León, Henry, Universidad Andrés Bello, Author
4. Saldarriaga-Córdoba, Mónica, Universidad Bernardo O’Higgins, Author

isolation and molecular characterization of aeromonas veronii and shewanella sp. from diseased koi carp (cyprinus carpio var. koi)

koi carp (cyprinus carpio var. koi) are ornamental fish of high economic value, widely demanded in the international aquarium trade due to their vibrant colors, patterns, and graceful swimming behavior. a recently deceased koi carp exhibiting swimming imbalance, abdominal swelling, and inappetence was submitted to our laboratory for diagnostic evaluation. additional mortalities and similar clinical signs were reported in other koi from the same pond.

necropsy revealed extensive scale loss and hemorrhages on the body surface, along with pronounced abdominal distension. internally, marked ascites and multifocal hemorrhages on visceral organs, particularly the heart, were observed. the swim bladder was deformed and exhibited nodular folding. samples from the liver, kidney, spleen, heart, and ascitic fluid were cultured on tryptic soy agar (tsa) and incubated at 22 °c for 24–48 hours. mucoid, cream-white colonies were isolated and purified.

preliminary identification was performed using maldi-tof ms, which revealed the presence of aeromonas veronii and shewanella decolorationis. genomic dna was extracted from the strains, and whole-genome sequencing was performed using the oxford nanopore platform. long-read libraries were generated from 400 ng of genomic dna using the oxford nanopore ligation sequencing kit (sqk-nbd114-24; oxford nanopore technologies, oxford, uk) following the manufacturer’s promethion protocol, without fragmentation. these libraries were loaded onto a promethion flow cell and sequenced for 24 hours using the p2 solo sequencer (ont). high-quality reads were assembled into contigs via de novo assembly using the unicycler hybrid assembly pipeline (v0.4.6).

the sequencing results confirmed a. veronii strain k-1, while shewanella sp. strain k-2 showed genomic features consistent with a potentially novel species based on taxonomic thresholds.

both aeromonas and shewanella genera are widely distributed in aquatic environments and are known to cause opportunistic infections in fish. their involvement in systemic hemorrhagic conditions and ascites is well documented, aligning with the pathological findings in this case. this case highlights the diagnostic value of integrating maldi-tof ms for rapid presumptive identification and whole genome sequencing for definitive species-level characterization. the complementary use of these techniques provided accurate, high-resolution taxonomic assignment and revealed the potential discovery of a previously uncharacterized shewanella species.

1. TAŞÇI, GÖRKEM, BURSA ULUDAG UNIVERSITY, Presenter
2. AJMI, NIHED, BURSA ULUDAG UNIVERSITY, Author
3. DUMAN, MUHAMMED, BURSA ULUDAG UNIVERSITY, Author
4. SATICIOĞLU, İZZET BURÇİN, BURSA ULUDAG UNIVERSITY, Author

Aliivibrio wodanis strain diversity and interaction with other ulcer-bacteria

Introduction

The bacteria most commonly associated with skin ulcers in Norwegian Atlantic salmon farmed in cold seawater are Moritella viscosa, Tenacibaculum finnmarkense, and Aliivibrio wodanis. While the two first are well recognized as bacterial pathogens with a strong skin ulcer association, the role of A. wodanis remains somewhat obscure. A. wodanis is often isolated from winter ulcers, together with other pathogens, but during recent years, has also been reported as the sole or major finding.

Thus, to address knowledge gaps, a collection of A. wodanis isolates form Norwegian aquaculture were investigated. We examined the overall population structure within A. wodanis in order to uncover possibly specific subpopulations, and described their physical and biochemical characteristics.

Methods

Phylogenetic recreation for examined A. wodanis isolates was based on two housekeeping genes. Isolates were also subjected to descriptive investigation of biochemical and physiological characteristics. In addition, co-culture experiments of A. wodanis isolates with M. viscosa and Tenacibaculum spp. were developed.

Results and Conclusion

The phylogenetic analysis revealed at least three distinct genetic lineages of A. wodanis associated with ulcer development in Atlantic salmon farmed in Norway. The selected isolates showed to some extent lineage-specific phenotypical characteristics, related to growth, morphological properties and co-culture behaviour. Co-cultivation experiments revealed that A. wodanis mediated growth inhibition on studied isolates of Tenacibaculum spp, and a variable inhibitory effect on M. viscosa.

1. Pettersen-Da, Synne, University of Bergen, Author
2. Roalkvam, Irene, NORWEGIAN VETERINARY INSTITUTE, Author
3. Olsen, Anne-Berit, NORWEGIAN VETERINARY INSTITUTE, Author
4. Colquhoun, Duncan, NORWEGIAN VETERINARY INSTITUTE, Author
5. Nilsen, Hanne Katrine, NORWEGIAN VETERINARY INSTITUTE, Presenter
6. Gulla, Snorre, NORWEGIAN VETERINARY INSTITUTE, Author

characterization of aeromonas salmonicida subsp. masoucida (asm) and streptococcus parauberis isolated from cultured platichthys stellatus (starry flounder) in south korea

aquaculture of starry flounder is increasingly important in south korea. however, as farming expands, mass mortality has risen significantly. while streptococcus parauberishas been recognized as a longstanding pathogen in korean aquaculture, recently, a. salmonicida subsp.masoucida(asm) has emerged as a novel pathogen in starry flounder aquaculture in south korea, posing a threat to aquaculture sustainability. globally, a. salmonicidais a well-known fish pathogen, occurring in both freshwater and marine environments. this study aims to investigate the phenotypic and molecular characteristics of asm and s. parauberisstrains isolated from farmed starry flounder. to identify the causative bacterial agents responsible for mass mortality in cultured platichthys stellatusin south korea, bacterial disease monitoring was conducted from april to december 2024. during this period, all isolated a. salmonicida ands. parauberis were identified through phylogenetic analysis based on 16s rrna gene sequences.all a. salmonicidaunderwent vapa-based analysis for subspecies classification. whole genome sequencing (wgs) was conducted to investigate virulence-associated genes and genomic characteristics. to evaluate pathogenicity, a standard curve between od₆₀₀ values and cfu/ml for both strains was established, followed by intraperitoneal challenge experiments. the ld₅₀ (lethal dose) was determined for a. salmonicidaad112405 to quantify its virulence. phenotypic, virulence gene, and antimicrobial resistance characteristics of all a. salmonicidaand s. parauberisisolates were examined for comparative analysis. the dominant species were identified as a. salmonicida, s. parauberis, and vibrio. scophthalmi. among 141 isolates; a. salmonicidawas presumed to be the primary causative agent of the mass mortality event. phylogenetic analysis using the vapa gene revealed that all a. salmonicidaisolates were reclassified as a. salmonicida subsp.masoucida. experimental infection with the representative strains ad112405 and ad112410 resulted in dose dependent mortality in challenged fish. the ld₅₀ of asm strain ad112405 was 3.93 × 10⁴cfu/fish. all asm exhibited beta hemolysis, 39 isolates had 9 of 14 virulence genes. one isolate carried an additional gene encoding serine protease. for s. parauberis, only gapcand hascwere detected in all isolates. serotyping revealed that all s. parauberisisolates belonged to serotype ⅰa. antimicrobial resistance gene screening showed that isolates from farms a and b carried erm(b) and tet(s). however, isolates from farm c did not carry the erm(b) gene. these results suggest that asm is an emerging pathogen responsible for recent mass mortality events in starry flounder aquaculture in korea.

1. Park, Ji-Hwan, PUKYONG NATIONAL UNIVERSITY, Presenter
2. Jung, Jae-Eun, PUKYONG NATIONAL UNIVERSITY, Author
3. Seo, Jeong-Min, PUKYONG NATIONAL UNIVERSITY, Author
4. Kim, Beom-Seok, PUKYONG NATIONAL UNIVERSITY, Author
5. Byun, Jeong-Won, PUKYONG NATIONAL UNIVERSITY, Author
6. Jeon, SO-YEON, PUKYONG NATIONAL UNIVERSITY, Author
7. Lim, Chae-Won, PUKYONG NATIONAL UNIVERSITY, Author
8. Jang, Seong-Won, PUKYONG NATIONAL UNIVERSITY, Author
9. Kim, Jong-Oh, PUKYONG NATIONAL UNIVERSITY, Author

Experimental infection approaches to investigate resistance to Bacterial Cold-Water Disease in rainbow trout

Bacterial cold-water disease (BCWD), also known as rainbow trout fry syndrome, is a devastating disease in freshwater salmonid farming worldwide. Flavobacterium psychrophilum, the causative agent, affects high commercial value salmonid species such as rainbow trout (Oncorhynchus mykiss) and Atlantic salmon (Salmo salar). In rainbow trout fry, which are particularly susceptible, the disease results in haemorrhagic septicaemia and high mortality. The treatment of BCWD exclusively relies on antibiotics, a practice that can foster the emergence of antibiotic-resistant bacteria potentially spreading to surrounding ecosystems, with implications for human, animal and environmental health. Genetic resistance to BCWD has been documented and selective breeding appears as an attractive strategy to reduce the impacts of outbreaks. Here, we developed experimental infection models in rainbow trout to enable phenotyping of the resistance to F. psychrophilum infections. The main objectives were to document the pathogenesis, identify critical factors affecting the outcome of infection and severity of the disease, and investigate the genetic and molecular basis of resistance. A comparative study of resistance phenotypes was first conducted using a collection of rainbow trout isogenic lines exhibiting contrasting susceptibilities to F. psychrophilum infection. We applied individual monitoring to follow the infection process using time-course determination of bacteraemia and host immune responses in blood. The results showed that disease resistance is associated with a better control of bacterial loads over time. Immune genes related to arginine metabolism, pathogen detection and clearance were overexpressed in blood of resistant fish compared to susceptible ones. Host-pathogen genotype interactions were also observed, with resistance levels varying depending on the bacterial strain used for infection. We also investigated the genetic basis of resistance in two French commercial rainbow trout populations by conducting a genome-wide association study (GWAS), using a standardized bath challenge model and high-density SNP genotyping. Genomic regions associated with resistance (QTLs) were detected in each population, providing valuable genomic information for selective breeding programs. Several candidate genes located within these regions were involved in immune signalling, inflammation, macrophages/neutrophils biology, and soluble factors important for antibacterial defences. This work paves the way for the production of more resistant animals through genetic selection and contributes to a better understanding of the mechanisms underlying resistance.

1. ROCHAT, Tatiana, INRAE, Presenter
2. LEE, Bo-Hyung, INRAE, Author
3. RIGAUDEAU, Dimitri, INRAE, Author
4. POUIL, Simon, INRAE, Author
5. SEGRET, Emilien, Viviers de Sarrance, Author
6. DESGRANGES, Alexandre, Les Aquaculteurs Bretons, Author
7. D’AMBROSIO, Jonathan, SYSAAF, Author
8. FRANCOIS, Yoannah, SYSAAF, Author
9. LALLIAS, Delphine, INRAE, Author
10. PHOCAS, Florence, INRAE, Author
11. BOUDINOT, Pierre, INRAE, Author
12. DUCHAUD, Eric, INRAE, Author

Environmental detection and the use of non-lethal sampling for diagnosis and detection of Renibacterium salmoninarum

Bacterial kidney disease (BKD) is a serious and notifiable disease caused by the gram-positive bacterium Renibacterium salmoninarum (RS). The disease is chronic and insidious, making it difficult to detect before the infection is widespread in the population. Effective detection of infected fish and populations is essential to manage the disease. In the autumn of 2024, BKD was detected at a land-based fish farm in Iceland that raises Atlantic salmon from roe to slaughter. The disease has been detected intermittently since 2017, and RS appears to persist in the facility. The facility uses both freshwater and seawater from several intakes and testing of roe have never yielded positive results. Repeated sampling of fish, water and the environment were performed from 25.09.2024 and throughout 2025 with the aims of determining which sample material and analysis methods will improve the sensitivity for detecting R. salmoninarum in Atlantic salmon with various stages of infection and disease, to what extent detection of bacteria in water corresponds to infection and disease status of the fish, and how the bacterium potentially enter, spreads and persists in the farming environment. 63 salmon from 6 fish groups and 8 tanks have been necropsied. Tissue was taken for RT-qPCR and histological analysis from the liver, gills, skin, spleen, kidney, and heart. PCR-analysis of homogenate of the head kidney and swabs from skin, cloaca, gills, and kidney were performed. Water and swabs from locations considered likely infection areas were analysed with RT-qPCR, with the aim of tracing a potential source of infection. So far, 56 swabs have been taken from the production environment, in addition to 22 water samples from 7 different locations. Preliminary results showed that 5 fish from the same tank and fish group had grossly observable multifocal white nodules in one or more tissues. RS was detected in most examined samples, including swabs from the gills, skin, and cloaca in all fish. Genetic material from RS was also detected in 16 fish without macroscopic changes consistent with BKD (Ct value: 18.04-32,91). Only effluent water from the tank containing diseased fish were positive for the bacterium (Ct value: 31.93). RS was widely detected in the production environment (27/56 swab samples, Ct value: 29.03-33.25). Further sampling, including bacterial culture of environmental samples is ongoing. We will present the preliminary results from this study, including tissue distribution of bacteria and lesions and correlation between fish, water and environmental detection of RS.

1. Østevik, Liv, NORWEGIAN UNIVERSITY OF LIFE SCIENCES, Presenter
2. Sandlund, Liv, Pharmaq Analytiq AS, Author
3. Ciani, Elia, Edmund Mach Foundation, Author
4. Laxdal, Bernharð, VETAQ, Author
5. Smáradóttir, Heiðdís, Samherji fiskeldi ehf, Author
6. Nørstebø, Simen, Norwegian Veterinary Institute, Author
7. Negash, Letemichael, NORWEGIAN UNIVERSITY OF LIFE SCIENCES, Author
8. Persson, David, NORWEGIAN UNIVERSITY OF LIFE SCIENCES, Author