Author: marina02

RPA based on-site molecular diagnostics for salmon virus infection diseases

Introduction

Infectious diseases in aquaculture cause substantial economic losses, demanding novel, effective and field-deployable solutions for early and rapid diagnostics. Recombinase Polymerase Amplification (RPA) technology offers tremendous potential for on-site molecular diagnostics in fish infectious disease control due to its isothermal amplification, speed, and minimal equipment needs. This study presents the development and evaluation of a novel RPA-based diagnostic system for rapid, on-farm detection of major viral pathogens in aquaculture.

Methodology

Our system integrates three key components: (1) A compact, battery-operated handheld device with Bluetooth connectivity; (2) Stabilized, freeze-dried reagents requiring no cold chain; and (3) A single-step, instrument-free DNA/RNA extraction protocol. A dedicated mobile application enables real-time reaction monitoring and result interpretation. System performance was evaluated against conventional RT-qPCR methods for detection of Infectious Pancreatic Necrosis Virus (IPNV), Piscine Myocarditis Virus (PMCV), and Piscine Orthoreovirus (PRV) in clinical samples.

Results

The entire diagnostic workflow from sample collection to the result takes approximately 30 minutes, with clear detection signals typically appearing within the first 10 minutes of the RPA reaction. Our system successfully identified all three target pathogens (IPNV, PMCV, PRV) in clinical samples at sensitive concentration levels comparable to those of conventional qPCR assays. In validation studies, our system demonstrated 100% concordance with laboratory based qPCR results, consistent with studies from similar RPA-based diagnostic platforms. Additional statistical data will be presented during the conference.

Conclusions

Our pilot study demonstrated the feasibility and effectiveness of RPA-based technology for rapid, on-site detection of viral pathogens in salmon aquaculture. Our immediate next steps involve expanding the panel of detectable salmonid pathogens and conducting more extensive field validation across diverse farming environments. Looking further ahead, we aim to adapt the system for other important aquatic species and diseases threats. We welcome collaborative opportunities to accelerate these developments and explore wider applications , including pathogens affecting warmwater fish.

1. GUO, YANWU, ANGENOVO AS, Presenter
2. Amit, Kumar Sharma, ANGENOVO AS, Author
3. Georgios, Spanos, Aquamedic AS, Author
4. Paul, J. Midtlyng, Aquamedic AS, Author

Establishment and phenotypic characterization of a new cell line derived from rainbow trout (Oncorhynchus mykiss) brain

Introduction

Cell lines are important tools for basic and applied research allowing robust in vitro studies under controlled experimental conditions. In particular, they are useful assets for development of disease control strategies as, for instance, a system for the production of viral particles to be used as an inactivated/attenuated vaccine for testing viral neutralisation efficacy of vaccines, or for infectiology (virulence studies), all within the remit of the 3Rs. Fish is one of the most diverse vertebrate groups and, as a consequence, the scientific community dedicated to their study is facing the difficulty of a multitude of experimental models. Salmonids are particularly important since they include the Atlantic salmon Salmo salar and the rainbow trout Oncorhynchus mykiss, two species central to a fast-growing worldwide fish farming industry.

Methodology

We describe here the isolation of RTB, a spontaneous immortalised cell line derived from the brain of the rainbow trout Oncorhynchus mykiss. This cell line grows as an adherent cell line in L15 supplemented with 10% bovine serum. The species identity was confirmed by PCR and sequencing. Host gene expression after stimulation with poly I:C or heat-killed Vibrio anguillarum was analysed by qPCR. The measure of viral production in the supernatant of infected RTB cells was also performed by qPCR. Cell transfection with a plasmid containing a fluorescent reporter system was done by electroporation with the Neon system. Transfection success was analysed by flow cytometry and quantification of fluorescence.

Results and discussion

RTB cells display cytoplasmic expansions when cultivated at low density and a fibroblastic-like morphology when closer to confluency. The cells sustain the replication and particle release of infectious pancreatic necrosis virus IPNV, viral haemorrhagic septicaemia virus VHSV and infectious hematopoietic necrosis virus IHNV. It responds to viral or bacterial PAMPs demonstrating functionality of both type I interferon and pro-inflammatory pathways. RTB can be transfected with 20% efficiency by electroporation, can be genetically modified and cloned. The level of expression of a number of markers specific for brain-related cell types is currently under investigation. A clonal version of RTB expressing the red fluorescent protein is currently under expansion and characterisation.

1. Peruzzi, Mathilde, INRAE, Author
2. Collins, Catherine, UCC, Author
3. Lavenot-Outin, Léa, INRAE, Author
4. Boudinot, Pierre, INRAE, Author
5. Pascale, Florentina, INRAE, Author
6. Dufour, Sylvie, MNHN, Author
7. Collet, Bertrand, INRAE, Presenter

Screening of five species of asymptomatic molluscs specimens sampled in coastal areas of Ria de Arousa, Galicia (NW Spain) reveals presence of Perkinsus olseni in two species of clams.

As part of a viruses and parasites surveillance in five shellfish coastal areas of Ría de Arousa (NW Spain), three types of samples were taken: water, sediments and mollusks, to elucidate if the presence of any known or unknown agent could explain the drop in production in certain locations. For this purpose, two types of procedures were employed, non-target (metagenomics and metatranscriptomics) and target (real time PCR [rtPCR]) techniques. In this communication, we present the results from rtPCR testing mollusks sampled in the first campaign (targeting herpes virus [OsHV-1], bonamia, marteilia and perkinsus [P. marinus and P. olseni]). In each location, two sites were selected: one with a history of production problems (wpp) and another without (wopp). A total of 56 individuals of three species of clam (Manila clam, Ruditapes philippinarum [14 indv]; banded carpet shell clam, Venerupis rhomboide [3 indv]; pullet carpet shell clam, V. corrugata [11 indv]), razor shell (Ensis arcuatus [14 indv]) and common European cockle (Cerastoderma edule [14 indv]) were tested. The hepatopancreas, the mantle or the entire vianda (the smallest individuals) were aseptically removed, and the DNA extracted from the tissues was subjected to rtPCR using procedures reported by the WOAH aquatic manual and other sources.

Only Perkinsus olseni was detected. Neither herpesvirus nor any of the other parasites were detected in any case. P. olseni was detected at high apparent prevalences in pullet carpet (63.6%) and Manila (28.6%) clams; it was not detected in banded carpet shell clam or razor shell and at a very low prevalence in cockle. Its distribution between wpp and wopp sites could not explain the differences in the production in four of the locations. In one case, the species was not present in the wpp site and did not carry the parasite in the wopp one. Nevertheless, additional and important information will come from the analysis of the water and sediment samples, and from the non-target study.

Financing: This study has been financed by i) project CALIMAR, Network REDEMAR, Consellería do Mar, Xunta de Galicia, through the European Maritime, Fisheries and Aquaculture Fund (EMFAF), ii) Red EpiMar (RED2022-134796-T), through MICIU/AEI/10.13039/501100011033, and European Regional Development Fund (ERDF), and iii) project EpiVir (PID2022-14200OB-10), through MICIU/AEI/10.13039/501100011033, and ERDF.

1. González-Rosales, Emma, ARCUS, UNIVERSIDADE DE SANTIAGO DE COMPOSTELA, Author
2. Olveira, José G., ARCUS, UNIVERSIDADE DE SANTIAGO DE COMPOSTELA, Author
3. López-Vázquez, Carmen, ARCUS, UNIVERSIDADE DE SANTIAGO DE COMPOSTELA, Author
4. Souto, Sandra, ARCUS,UNIVERSIDADE DE SANTIAGO DE COMPOSTELA, Author
5. Dopazo, Carlos, ARCUS, UNIVERSIDADE DE SANTIAGO DE COMPOSTELA, Presenter

Partial Validation of Two Real-Time PCR Detection Methods for Decapod Iridescent Virus

The Decapod Iridescent Virus (DIV1) is a double-stranded DNA virus. According to the criteria set by the World Organisation for Animal Health (WOAH), there are eight species susceptible to DIV1 infection, including the whiteleg shrimp, ridgetail prawn, red claw crayfish, red swamp crawfish, giant river prawn, Oriental river prawn, kuruma prawn, and swimming crab. This is the latest crustacean disease listed by WOAH. To better control the transboundary spread of this virus and support WOAH reporting requirements, we conducted the first and second phase validations of two real-time PCR (QPCR) detection methods listed in the WOAH Aquatic Animal Manuals, following the principles and methods for validating diagnostic assays. We analyzed the 100% detection limit (LOD), diagnostic sensitivity (DSe), and diagnostic specificity (DSp) of these methods. Using the ATPase Nested PCR method as the gold standard for calculations, the results showed that the LOD100 for the MCP gene QPCR and ATPase gene QPCR methods were 12.75 copies/20 μL QPCR reaction and 15.26 copies/20 μL QPCR reaction, respectively. Both methods exhibited the same diagnostic sensitivity (DSe) and diagnostic specificity (DSp) of 88.43% (95% CI: 80.8%, 91.4%) and 91.36% (95% CI: 90.8%, 96.0%). Their agreement with the gold standard tests was also very high, with Kappa values of 0.885 and 0.876, respectively. The results of this study will further provide epidemiological monitoring and assessment tools, such as true prevalence rates, positive predictive values, and negative predictive values, along with the formulation of relevant disease prevention policies for DIV1.

1. LU, YI PING, NATIONAL PINGTUNG UNIVERSITY OF SCIENCE AND TECHNOLOGY, Presenter
2. Lin, Tzu En, Veterinary Research Institute, Council of Agriculture, Author
3. Lin, Yu Ju, Veterinary Research Institute, Council of Agriculture, Author
4. Cheng, Ming Chu, National Pingtung University of Science and Technology, Author

Development of Cyprinid herpesvirus-2 detection with CRISPR-Cas 12a system

Cyprinid herpesvirus-2 (CyHV-2), a member of the Alloherpesviridae family, is a significant pathogen impacting ornamental goldfish (Carassius auratus), especially in low water temperatures (<27°C). Due to the potential for latent infections and international transmission, a rapid and specific diagnostic tool is essential. This study reports the development of a detection system combining Recombinase Polymerase Amplification (RPA) and CRISPR-Cas12a, targeting the highly specific marker B region of the CyHV-2 genome. We designed six candidate crRNAs based on the PAM sequence at marker B, of which CmB2 and CmB5 showed the most promising performance. RPA primers were optimized for temperature and incubation time, with 35°C for 5 minutes yielding the best amplification results. The CRISPR-Cas12a system, coupled with RPA, achieved a detection limit of 10⁴ viral copies/µL using CmB2 at optimal conditions. Although CmB5 was functional at room temperature (25°C), it showed delayed signal development and lower sensitivity, indicating that 35°C remains preferable for rapid screening. Importantly, no cross-reactivity was observed with other major aquatic viruses (KHV, CEV, SDDV, ISKNV, iridovirus) or common bacterial pathogens (e.g., Plesiomonas spp., Edwardsiella ictaluri, Aeromonas veronii, Flavobacterium columnare). This specificity underscores the reliability of our system in differentiating CyHV-2 from other infectious agents, including closely related alloherpesviruses. In summary, the integration of RPA and CRISPR-Cas12a provides a sensitive, specific, and rapid diagnostic platform for CyHV-2 detection. The system’s simplicity, minimal equipment requirements, and ability to deliver visual results within minutes support its potential application in aquaculture diagnostics and on-site pathogen surveillance.

1. RODKHUM, CHANNARONG, Center of Excellence in Fish Infectious Diseases (CE FID),FACULTY OF VETERINARY SCIENCE, CHULALONGKORN UNIVERSITY, THAILAND, Presenter
2. Pirarat, Nopadon, Department of Pathology, Faculty of Veterinary Science, Chulalongkorn University, Thailand, Author
3. Panthukumphol, Naphat, Center of Excellence in Fish Infectious Diseases (CE FID),FACULTY OF VETERINARY SCIENCE, CHULALONGKORN UNIVERSITY, THAILAND, Author
4. Khianchaikhan, Kamolwan, Center of Excellence in Fish Infectious Diseases (CE FID),FACULTY OF VETERINARY SCIENCE, CHULALONGKORN UNIVERSITY, THAILAND, Author

Development and validation of a multiplex RT-PCR assay for the simultaneous detection of three major viral pathogens in salmonids, IHNV, ISAV and SAV

Infectious hematopoietic necrosis virus (IHNV), infectious salmon anaemia virus (ISAV), and salmon alphavirus (SAV) are significant pathogens responsible for severe infections in salmonid species and are classified as notifiable diseases by the World Organization for Animal Health (WOAH). This study focused on the selection of optimal primer pairs that target highly conserved regions within the genomes of each virus, emphasizing specificity and broad genotype coverage, which were subsequently assessed through single PCR assays. The limit of detection (LOD) for each single PCR was determined to be 1.0×10⁰ copies/µL for IHNV, 1.0×10¹ copies/µL for ISAV, and 1.0×10² copies/µL for SAV. A multiplex RT-PCR assay was developed to simultaneously detect the three pathogenic salmonid viruses alongside the β-actin gene, which served to validate RNA isolation and cDNA synthesis. The multiplex PCR was optimized to include 0.3 mM dNTPs, 0.5 mM betaine, and GC Melt solution, with an annealing temperature set at 60°C. The LOD for the multiplex PCR was established at 1.0×10³ copies/µL for IHNV, 1.0×10² copies/µL for both SAV and ISAV, with a calculated LOD_95% of 7.4×10² copies/µL for all viruses. Importantly, the multiplex PCR did not yield non-specific amplification in eight different fish cell lines and four bacterial isolates, with further validation using field samples from aquaculture farms showing no false positives, and robustness evaluations demonstrating consistent and reliable performance across various PCR reagents.

1. Ko, Sungjae, Department of Aquatic Life Medicine, Gangneung-Wonju National University, Gangneung 25457, Republic of Korea, Presenter
2. Seong, So min, Department of Aquatic Life Medicine, Gangneung-Wonju National University, Gangneung 25457, Republic of Korea, Author
3. Kim, Young Chul, Department of Aquatic Life Medicine, Gangneung-Wonju National University, Gangneung 25457, Republic of Korea, Author
4. Kim, Kwang Il, Department of Aquatic Life Medicine, Pukyong National University, Busan 48513, Republic of Korea, Author
5. Kwon, Mun Gyeong, Aquatic Disease Control Division, National Fishery Products Quality Management Service (NFQS), Republic of Korea, Author
6. Jeong, Ji-Min, Aquatic Disease Control Division, National Fishery Products Quality Management Service (NFQS), Republic of Korea, Author
7. Hong, Suhee, Department of Aquatic Life Medicine, Gangneung-Wonju National University, Gangneung 25457, Republic of Korea, Author

Development of SYBR Green-based real-time PCR assays for detection of infectious haematopoietic necrosis virus (IHNV)

The risk of introducing unreported genotypes of infectious haematopoietic necrosis virus (IHNV) into South Korea has increased due to the global expansion of aquaculture and the international movement of live fish and eyed eggs. Although real-time PCR using TaqMan probes is currently recommended by the World Organisation for Animal Health, questionable amplification results have been observed in certain viral strains due to single nucleotide mismatches in the probe-binding region, potentially reducing the assay’s diagnostic performance. This study aimed to develop and validate a SYBR Green-based real-time PCR assay for the detection of various IHNV genotypes (E, U, M, JN and JS), providing a cost-effective and sensitive alternative to existing probe-based real-time PCR assay. Primers were designed to target conserved regions of IHNV nucleoprotein (N) gene using in silico analysis to ensure high specificity and minimal dimer formation. Assay optimization was conducted using cDNA synthesized from uninfected salmonid tissues, bacterial and virus and plasmid standards. The optimized assay demonstrated excellent analytical sensitivity and specificity, achieving a 95% limit of detection (LoD₉₅%) of 16.92 copies/reaction (95% CI: 14.08–21.82). Diagnostic validation using experimentally infected samples showed 100% diagnostic sensitivity and 100% diagnostic specificity. Repeatability testing across experimenters and time points yielded a coefficient of variation below 5%, indicating high reproducibility in accordance with the FDA Bioanalytical Method Validation Guidelines. In conclusion, the SYBR Green-based real-time PCR assay developed in this study offers a reliable, scalable, and cost-efficient diagnostic tool for the early detection of IHNV. Its high analytical and diagnostic performance may support its application in routine screening, outbreak surveillance, and quarantine programs, particularly in resource-limited aquaculture settings, thereby contributing to improved biosecurity and sustainable fish health management.

1. Song, Eunsu, Aquatic Life Medicine, Gangneung-Wonju National University, Gangneung 25457, Republic of Korea, Presenter
2. Ko, Sungjae, Aquatic Life Medicine, Gangneung-Wonju National University, Gangneung 25457, Republic of Korea, Author
3. Kim, Young Chul, Aquatic Life Medicine, Gangneung-Wonju National University, Gangneung 25457, Republic of Korea, Author
4. Kwon, Mun Gyeong, Aquatic Disease Control Division, National Fishery Products Quality Management Service (NFQS), Republic of Korea, Author
5. Jeong, Ji-Min, Aquatic Disease Control Division, National Fishery Products Quality Management Service (NFQS), Republic of Korea, Author
6. Hong, Suhee, Aquatic Life Medicine, Gangneung-Wonju National University, Gangneung 25457, Republic of Korea, Author

Development of SYBR Green-based real-time PCR assays for detection of salmonid alphavirus (SAV)

With the intensification of international trade and diversification of salmonid aquaculture, the risk of introducing unreported genotypes of Salmonid alphavirus (SAV) into South Korea has substantially increased. Although South Korea was officially declared free from SAV in 2023, the ongoing emergence of diverse genotypes highlights the urgent need for robust diagnostic tools to ensure continued biosecurity. Existing real-time PCR assays recommended by the World Organisation for Animal Health (WOAH) have been validated only for SAV genotype 3, raising concerns regarding their diagnostic breadth and sensitivity for other genotypes. Moreover, most SAV diagnostic assays have been analytically validated using synthetic constructs or laboratory strains, with limited evaluation using field or experimentally infected tissue samples. To address this gap, we developed and validated a SYBR Green-based real-time PCR assay targeting conserved regions of the nsP1 gene to detect SAV genotypes 1, 2, 5 and 6. Positive tissue samples were obtained through experimental infection of rainbow trout with different SAV genotypes and used to assess diagnostic performance. The assay demonstrated a 95% limit of detection (LoD₉₅_%) of 21.73 copies/reaction (95% CI: 17.56–29.54). Diagnostic sensitivity and specificity were both confirmed to be 100% based on comparison with WOAH-recommended assays. Repeatability testing revealed a coefficient of variation (CV) below 5% across experimenters and time points, underscoring the assay’s reproducibility and robustness. Clear amplification curves and single melting peaks were observed, with no evidence of nonspecific signals. In conclusion, the validated SYBR Green-based real-time PCR assay provides a sensitive, specific, and affordable platform for the detection of diverse SAV genotypes. Its high reproducibility and ability to detect various SAV genotypes make it suitable for routine diagnostics, outbreak surveillance and biosecurity monitoring in salmonid aquaculture.

1. Song, Eunsu, Aquatic Life Medicine, Gangneung-Wonju National University, Gangneung 25457, Republic of Korea, Presenter
2. Ko, Sungjae, Aquatic Life Medicine, Gangneung-Wonju National University, Gangneung 25457, Republic of Korea, Author
3. Kwon, Mun Gyeong, Aquatic Disease Control Division, National Fishery Products Quality Management Service (NFQS), Republic of Korea, Author
4. Jeong, Ji-Min, Aquatic Disease Control Division, National Fishery Products Quality Management Service (NFQS), Republic of Korea, Author
5. Hong, Suhee, Aquatic Life Medicine, Gangneung-Wonju National University, Gangneung 25457, Republic of Korea, Author

Ontario Ministry of Natural Resources provincial hatchery fish health monitoring status in Ontario, Canada (2022–2025)

Introduction. Between April 1, 2022, and March 31, 2025, the Ontario Ministry of Natural Resources (MNR) undertook a comprehensive fish health monitoring initiative in collaboration with the Animal Health Laboratory at the University of Guelph. The primary objective was to monitor disease presence in cultured fish populations, thereby supporting the production of disease-free fish for stocking purposes, enhancing the health of wild fish populations, creating and maintaining fishing opportunities, and contributing to biodiversity restoration in Ontario’s aquatic ecosystems. Additional secondary objectives included investigating mortality events for pathogen involvement, screening wild fish used for spawning purposes and assessing fish held in quarantine.

Ontario operates nine (9) fish culture stations located across the province. Approximately eight million fish are stocked annually into over 1200 water bodies.

Fish are reared for stocking and maintaining captive broodstock. Species include, but are not limited to:

• Atlantic salmon (Salmo salar)
• Brook trout (Salvelinus fontinalis)
• Brown trout (Salmo trutta)
• Chinook salmon (Oncorhynchus tshawytscha)
• Lake trout (Salvelinus namaycush)
• Rainbow trout (Oncorhynchus mykiss)
• Splake (Salvelinus namaycush x Salvelinus fontinalis)
• Lake whitefish (Coregonus clupeaformis)
• Walleye (Sander vitreus)

Methodology. Routine fish health assessments were conducted in the nine MNR fish culture facilities, following standardized sampling methodology. Annually, 165 fish were sampled per facility, totalling 6,930 fish over the study period. Samples were pooled (4 fish per pool) and tested as follows:

• Necropsy: 1,697 pools
• Bacteriological culture: 4,705 pools
• Bacterial and viral PCR testing: 9,512 pools

Results and discussion:

• PCR Results: Of the 9,512 PCR tests conducted, only 87 (0.91%) were positive for target pathogens, with Renibacterium salmoninarum being the most common (55 detections). No highly impactful pathogens such as IHNV, ISAV, and Myxobolus cerebralis were found.
• Bacterial Cultures: Roughly half of the samples (49.04%) showed no bacterial growth, and an additional 27.4% showed no known bacterial pathogens, suggesting a predominantly healthy microbial environment. The most frequently isolated bacteria were environmental or opportunistic, with known pathogenic species like F. psychrophilum and A. salmonicida present but at low prevalence.

Conclusion

The fish health monitoring data from 2022 to 2025 reflect a positive status for Ontario’s fish culture stations:

• Low detection of pathogenic organisms
• Absence of major notifiable viral pathogens

These findings underscore the effectiveness of current biosecurity, surveillance, and health management protocols within Ontario’s Fish Culture Section. Continued vigilance through systematic monitoring will be essential to preserve fish health, prevent outbreaks, and support long-term ecosystem and biodiversity goals.

1. CAI, HUGH, University of Guelph, Presenter
2. Hobden, Kerry, Ontario Ministry of Natural Resources, Author
3. Addison, Peter, Ontario Ministry of Natural Resources, Author
4. Kellendonk, Calvin, University of Guelph, Author
5. Al-Hussinee, Lowia, University of Guelph, Author
6. Yu, Qiumei, University of Guelph, Author
7. Snyman, Heindrich, University of Guelph, Author
8. Bennoit, Nathan, University of Guelph, Author
9. Ledger, Lisa, University of Guelph, Author
10. Brock, Tanya, University of Guelph, Author
11. Munevar, Fernando, University of Guelph, Author
12. Nelson_Smikle, Pauline, University of Guelph, Author

Bayesian evaluation of sensitivity and specificity of three PCR-based assays to detect Aphanomyces astaci in crayfish

Introduction

Aphanomyces astaci, the pathogen responsible for crayfish plague, is causing decline and extinction of native freshwater crayfish populations in Switzerland and Europe. Therefore, crayfish plague is classified as reportable disease in Switzerland and is listed by the WOAH (World Organisation for Animal Health). In recent years, various molecular methods have been developed to diagnose the presence of A. astaci. However, there is no gold-standard available. To develop effective monitoring and control strategies, it is essential to understand the sensitivity and specificity of the commonly used diagnostic tests.

Material and methods

We used Bayesian latent class modeling to compare sensitivity and specificity of a conventional PCR protocol (Oidtmann et al., 2006) with minor modifications and of two qPCR protocols (Vrålstad et al., 2009, modified by Strand et al., 2013; Strand et al., 2023) using DNA extracted from 365 individual crayfish collected from 16 wild populations with varying A. astaci prevalence and pathogen abundance across Switzerland.

Results and Discussion

Among the tested crayfish samples, the qPCR protocol by Vrålstad et al. (2009, modified by Strand et al., 2013) yielded the highest number of positive individuals but showed the most inconsistencies within technical replicates. The modified conventional PCR protocol (Oidtmann et al., 2006) showed the lowest number of positive results. The qPCR protocol by Strand et al. (2023) demonstrated higher consistency within technical replicates and detected more positive individuals overall than Oidtmann et al. (2006). The modelling results are still pending.

1. Hermanns, Julius, Institute for Fish and Wildlife Health, Vetsuisse Faculty, University Bern, Presenter
2. Jemmi, Eliane, Institute for Fish and Wildlife Health, Vetsuisse Faculty, University Bern, Author
3. Pisano, Simone Roberto Rolando, Institute for Fish and Wildlife Health, Vetsuisse Faculty, University Bern, Author
4. Delalay, Gary, Institute for Fish and Wildlife Health, Vetsuisse Faculty, University Bern, Author
5. Diserens, Nicolas, Institute for Fish and Wildlife Health, Vetsuisse Faculty, University Bern, Author
6. Lüthi, Ramona, Institute for Fish and Wildlife Health, Vetsuisse Faculty, University Bern, Author
7. Schmidt-Posthaus, Heike, Institute for Fish and Wildlife Health, Vetsuisse Faculty, University Bern, Author