Author: marina02

Single-nuclei RNA Sequencing Reveals the Underpinnings of the Variation in Sea Lice Resistance Among Host Salmonid Species

Sea lice pose one of the greatest threats to wild and farmed populations of Atlantic Salmon (Salmo salar). However, the resistance to this ectoparasite varies markedly among salmonid species, with certain Pacific salmonids (Oncorhynchus spp.) demonstrating remarkable innate immunity. Our previous research demonstrated that the epithelial hyperplasia employed by the resistant coho salmon (O. kisutch) to kill or dislodge attached lice over the course of several days arises from a distinct but coordinated response of three layers of keratinocytes. In contrast, Atlantic salmon exhibit signs of greater immunomodulation, which may underlie their heightened susceptibility to sea lice. Here we study two additional Oncorhynchus species with contrasting susceptibilities to sea lice—the highly resistant pink salmon (O. gorbuscha) and the vulnerable chum salmon (O. keta)—to assess the consistency of the molecular mechanisms driving resistance and susceptibility across species.

We used single-nuclei RNA sequencing to characterize the cell type-specific transcriptomic response within the skin of pink salmon and chum salmon. Juvenile fish were exposed to sea lice (Lepeophtheirus salmonis) copepodids, and fin and skin samples were collected from control and infected individuals at the point of lice attachment at 12h, 36h, 48h, and 60h post-exposure. Nuclei were isolated and then barcoded using Chromium (10X Genomics), sequenced with Illumina, and analysed with STAR and Seurat.

Chum salmon keratinocytes upregulated several of the same genes previously identified in coho salmon as part of their response to sea lice; however, this upregulation generally occurred at a later time point (60h), suggesting a delayed recognition or impaired early response to the parasite. In contrast, pink salmon mounted a strong immune response as at the earliest observed infection timepoint (12h), including the upregulation of several coho-associated genes. Notably, pink salmon also upregulated numerous immune-related genes distinct from those employed by coho salmon, particularly within T cells and macrophages, indicating a unique molecular basis for resistance in each of these species. These findings support the hypothesis that rapid immune activation underlies the swift rejection of lice in pink salmon, in contrast to the slower, epithelial hyperplasia–mediated response observed in coho salmon. Our identification of genes associated with pink salmon’s resistance to L. salmonis offers insights into the evolutionary predictability of parasite resistance and highlights molecular targets to enhance sea lice resistance in cultured Atlantic salmon.

1. Salisbury, Sarah J, University of Exeter, Presenter
2. Ruiz Daniels, Rose, University of Stirling, Author
3. Monaghan, Sean J, University of Stirling, Author
4. Bron, James, University of Stirling, Author
5. McGowan, Michael, University of Stirling, Author
6. Paula, Villamayor, University of Santiago de Compostela, Author
7. Gervais, Ophelie, The Roslin Institute, University of Edinburgh, Author
8. Sveen, Lene, Nofima, Author
9. Fast, Mark D, Atlantic Veterinary College, University of Prince Edward Island, Author
10. Houston, Ross D, Benchmark Genetics, Author
11. Robinson, Nicholas, Nofima, Deakin University, Author
12. Robledo, Diego, The Roslin Institute, University of Santiago de Compostela University of Edinburgh, Author

Investigation of proteins identified in the secretory and excretory products (SEPs) of the infectious copepodid stage of the salmon louse Lepeophtheirus salmonis

Background: The salmon louse (Lepeophtheirus salmonis (Krøyer, 1837)) is a caligid copepod ectoparasite of salmonids that feeds upon the blood, mucus, and skin of its hosts. While later life stages are known to secrete virulence factors that dampen host immune responses and facilitate feeding and survival, the functional protein components of secretory and excretory products (SEPs) produced by copepodids, the initial infectious stage of L. salmonis, remain uncharacterized.

Methodology: L. salmonis copepodids were hatched from egg strings and incubated at 10°C until 7 days post-hatch. Copepodid batches (n=4 replicates) were then incubated for three hours at 10°C to allow SEP production. Adult males and females (n=2 replicates) were incubated separately at densities of three or two lice mL⁻¹, respectively, under the same conditions for comparison. SEPs were filtered, precipitated, digested with trypsin, and subjected to LC-MS/MS analysis to determine protein composition. Proteins were identified by searching LC-MS/MS data against an L. salmonis database and further analyzed in silico with SignalP and InterPro.

Results: A total of 433 distinct L. salmonis proteins were detected across copepodid samples (mean 95.5 ± 146.74 (1 S.D.)), while adult samples contained 117 proteins (mean 56 ± 12.70). Investigation of signal peptides revealed 164 copepodid and 69 adult proteins were secretory in nature. Among adult samples, 31 secreted proteins were unique to females and 10 to males. Across treatments, adult and copepodid SEPs shared 21 secreted proteins, including 8 proteases, 2 protease inhibitors, and 2 uncharacterized proteins. Of these proteins with GO annotations for biological process and cellular component, 75% were involved in proteolysis and 50% were localized extracellularly. However, adult and copepodid secretory proteins differed considerably in composition and cellular localization, with distinct proteases, protease inhibitors, and proteins implicated in immune regulation. Notably, 67% of the 9 secreted proteins with GO terms found only in adults were extracellular, compared to 30.7% of 52 copepodid-specific secreted proteins. Additionally, copepodid and adult secretions contained 23 and 4 unique uncharacterized proteins, respectively.

Conclusions: Analysis of L. salmonis copepodid secretions reveals a diverse array of proteins putatively involved in the facilitation of parasitism. The characterization of these proteins provides novel insights into early host-parasite interactions and identifies potential drug and vaccine targets, warranting further investigation through in silico analysis and in situ hybridization.

1. Dindial, Alexander, University of Stirling, Presenter
2. Monaghan, Sean, University of Stirling, Author
3. Haywood, Jay, University of Stirling, Author
4. McLean, Kevin, Moredun Research Institute, Author
5. Androscuk, Dorota, Moredun Research Institute, Author
6. Thompson, Kim, Moredun Research Institute, Author
7. Roy, William, Moredun Scientific, Author
8. Bron, James, University of Stirling, Author

Oral delivery of bioencapsulated virus-like particles confers effective protection against nervous necrosis virus in farmed fish

Introduction

Nervous necrosis virus (NNV), a member of the Nodaviridae family, infects over 170 fish species and causes significant economic losses in the aquaculture industry. Current injectable vaccines are unsuitable for large-scale application due to handling stress and labor-intensive administration. Therefore, oral vaccination via feed offers a promising alternative, providing non-invasive, cost-effective approach to mass immunization. Among oral vaccine platforms, virus-like particles (VLPs) are self-assembled viral capsid proteins without genetic material, closely mimic virions and are highly immunogenic, making them ideal candidates for oral delivery.

Methodology

In the study, recombinant E.coli cells were engineered to express NNV viral capsid proteins in the VLP form inside. Further, the bacterial cells were either inactivated or lysed without compromising the capsid structure and then incorporated into fish feed for oral vaccination. The immunogenicity and protective efficacy of both inactivated whole-cell and lysed E.coli containing NNV VLPs were evaluated in Asian seabass (ASB), and compared with purified VLPs administered via oral or intraperitoneal (IP) routes. Protective efficacy was also validated in orange-spotted grouper (OSG) following NNV challenge.

Results 

The results showed that intraperitoneal immunization with lysed E. coli expressing NNV VLPs induced the highest virus neutralizing antibody titers against RGNNV in ASB. Among the orally vaccinated groups, fish fed with inactivated whole-cell E. coli expressing NNV VLPs exhibited significantly higher NNV-specific IgM levels and virus neutralizing antibody titer compared to those immunized with lysed E. coli or purified NNV-VLPs. Disease challenge trials revealed a 3-log reduction in brain viral loads in ASB orally vaccinated with either lysed or whole cell E. coli expressing NNV VLPs. This protective effect was further validated in orange-spotted grouper, a species highly susceptible to NNV, which showed 100% survival following oral vaccination.

Conclusions

Our findings demonstrate that oral delivery of bioencapsulated VLPs as a safe, scalable, and effective oral vaccine platform for farmed fish. This approach eliminating the need for protein purification, representing a significant advancement in fish vaccinology.

1. PRABAKARAN, MOOKKAN, Temasek Life Sciences Laboratory, 1 Research Link, National University of Singapore, Singapore 117604, Presenter
2. Yang, Daiwen, Department of Biological Sciences, National University of Singapore, 14 Science Drive 4, Singapore, Author
3. Janlin, Chan, Temasek Life Sciences Laboratory, 1 Research Link, National University of Singapore, Singapore 117604, Author
4. Hui Yee, Hong, Department of Biological Sciences, National University of Singapore, 14 Science Drive 4, Singapore, Author
5. Lee, Carmen, Temasek Life Sciences Laboratory, 1 Research Link, National University of Singapore, Singapore 117604, Author

 Prime-boost vaccination with recombinant lice antigens confers protection against experimental Lepeophtheirus salmonis infestation in Atlantic salmon (Salmo salar L.)

Koestan Gadan, Therese Corneliussen and Øystein Evensen 

Norwegian University of Life Sciences Faculty of Veterinary Medicine, P.O. Box 5003, N-1432 Ås, Norway,

Sea lice, particularly Lepeophtheirus salmonis, are ectoparasitic copepods that present a major challenge to salmon aquaculture worldwide. Infestations compromise fish welfare, reduce growth rates, and increase mortality, leading to significant economic losses and raising concerns over sustainability. Traditionally, control of sea lice has relied heavily on chemotherapeutic agents. However, widespread and repeated use has driven the emergence of resistant lice populations, diminished the effectiveness of these treatments necessitates the development of alternative strategies.

Vaccination represents a sustainable, environmentally friendly approach to sea lice control. While several antigen candidates have been investigated, including those derived from the parasite’s digestive and structural proteins, field efficacy remains variable. Moreover, sea lice infestation itself is known to modulate host immune responses, potentially influencing susceptibility to secondary infections and reducing the efficacy of conventional vaccines.

In this study, we evaluated the efficacy and longevity of a prime-boost vaccination strategy utilizing two chimeric recombinant antigens, TT-P0 and P0-my32, derived from conserved proteins of L. salmonis. These antigens were formulated in an oil-based adjuvant to enhance immunogenicity Fish were experimentally challenged with copepodids at 2, 5 and 11 months post prime vaccination.

Vaccinated fish exhibited a statistically significant reduction in lice burdens across all challenge time points compared to unvaccinated controls. This protective effect was associated with elevated levels of circulating antigen-specific antibodies, although a direct link between antibodies and protection is elusive. These findings support the potential of chimeric antigen-based vaccines as an effective tool for integrated sea lice management in salmon aquaculture.

1. GADAN, KOESTAN, NMBU, Presenter
2. Øystein, Evensen, NMBU, Author
3. Corneliussen, Therese, NMBU, Author

Efficacy of an autogenous vaccine against Tenacibaculum maritimum in European Sea bass (Dicentrarchus labrax)

Introduction

Tenacibaculum maritimum is a significant pathogen in European seabass (Dicentrarchus labrax) farming, causing ulcerative disease known as tenacibaculosis. The infection often results in severe skin lesions, gill necrosis, and high mortality, especially during warmer months. No commercial vaccines currently exist for seabass, and control is largely dependent on good aquaculture practices.

Methodology

T. maritimum was isolated from symptomatic seabass on Adriatic Sea fish farms. Colonies were grown on FMM agar, identified via biochemical profiling (API 20E) and 16S rRNA sequencing. A formalin-inactivated whole-cell vaccine (FMC) was prepared by culturing biomass in marine broth, followed by ultrasonic homogenization and formaldehyde inactivation. Sterility and safety were checked. Juvenile seabass were vaccinated either by immersion (2 minutes in a 1:9 diluted vaccine; 3.75×10⁶ CFU/mL) or intraperitoneal injection (IP; 0.2 mL/fish; 6.0×10⁷ CFU/mL). Control groups were injected with PBS. Vaccine efficacy was evaluated through expression analysis of immune-related genes in gills, head kidney, and spleen using qPCR. Target genes included those related to humoral and acquired immunity (IgT, p35), and innate immunity (IL-1β, IL-10). Sera were collected 30 days post-vaccination and tested for specific IgM antibodies against T. maritimum using ELISA. Fish were then challenged by immersion or IP injection with live T. maritimum (7.2×10⁷ CFU/mL). Relative Percent of Survival (RPS) was calculated using formula: RPS = (1 – mortality in vaccinated group / mortality in control group) × 100%.

Results

No signs of disease or mortality were observed in any group during the 35 days post-vaccination. All tested genes showed upregulated expression following vaccination. ELISA revealed that immersion-vaccinated fish had IgM levels comparable to controls, whereas IP-vaccinated fish exhibited significantly higher titers. Following challenge, IP-vaccinated fish achieved an RPS of 65%, while immersion-vaccinated fish showed complete protection (RPS = 100%). Infected fish displayed typical clinical signs of tenacibaculosis, including oral and fin erosions.

Conclusions

The autogenous vaccine conferred significant protection against tenacibaculosis when administered either by immersion or intraperitoneal (IP) injection. Vaccination represents a promising strategy for preventing the disease in juvenile sea bass. However, further field trials are necessary to confirm these laboratory findings under commercial farming conditions.

1. ZUPIČIĆ, IVANA GIOVANNA, CROATIAN VETERINARY INSTITUTE, CROATIA, Presenter
2. ORAIĆ, DRAŽEN, CROATIAN VETERINARY INSTITUTE, CROATIA, Author
3. ALFIER, MATEA, CROATIAN VETERINARY INSTITUTE, CROATIA, Author
4. GRBIN, DOROTEA, CROATIAN VETERINARY INSTITUTE, CROATIA, Author
5. VOLPATTI, DONATELLA, UNIVERSITY OF UDINE, ITALY, Author
6. GALLEOTTI, MARCO, UNIVERSITY OF UDINE, ITALY, Author
7. ZRNČIĆ, SNJEŽANA, CROATIAN VETERINARY INSTITUTE, CROATIA, Author

The effects of oral Lactobacillus delbrueckii and Lacticaseibacillus rhamnosus supplement on the pharmacokinetics of florfenicol in Nile tilapia (Oreochromis niloticus): implications on reduced therapeutic efficacy

Dietary supplementation with probiotics such as lactic acid bacteria (LAB) has gained increasing popularity in aquaculture due to their beneficial effects on fish growth performance and immunity. Probiotic supplementation enhances disease resistance in fish; however, antibiotics are sometimes still required to control bacterial infections. Despite this, few studies have investigated whether probiotic use may alter drug behavior within the body. This study examined the effects of LAB supplementation on the pharmacokinetics (PK) of florfenicol (FF) in Nile tilapia (Oreochromis niloticus) reared at 25°C. Healthy fish (300 ± 50 g body weight) were orally administered a mixture of Lactobacillus delbrueckii and Lacticaseibacillus rhamnosus (1 × 10⁹ CFU/fish/day) for 10 consecutive days, followed by a single oral dose of FF (10 mg/kg). Serial blood samples were collected from each fish between 0.25 and 72 hours post-administration, and serum FF concentrations were measured using HPLC-FLD. PK parameters were calculated using a two-compartment model, and the optimal dosage was estimated using a pharmacokinetic-pharmacodynamic (PK-PD) approach. Additionally, mRNA levels of cytochrome P450 enzymes (cyp1A and cyp3A) in the liver and proximal intestine were analyzed by RT-qPCR in fish sacrificed at 72 hours post-FF administration to assess potential changes in drug metabolism. The results showed that LAB supplementation reduced the elimination half-life (t₁/₂β) of FF from 16.7 to 14.1 hours. The maximum serum concentration (Cmax) was 48% lower in LAB-treated fish (9.09 ± 1.62 µg/mL) compared to untreated fish (17.51 ± 0.99 µg/mL). The apparent clearance (CL/F) in LAB-treated fish was approximately double that of the control group (0.11 vs. 0.052 L/kg/h), and the calculated optimal dosage at a minimum inhibitory concentration (MIC) of 2 µg/mL was likewise doubled (11.07 vs. 5.97 mg/kg/day). Furthermore, RT-qPCR analysis revealed that LAB administration upregulated cyp3A expression in the liver and increased cyp1A transcription in the proximal intestine. In conclusion, LAB pretreatment accelerated FF elimination and reduced systemic drug exposure in Nile tilapia, indicating that higher FF doses may be required to achieve therapeutic efficacy in probiotic-supplemented fish. Whether or not the upregulated cyp genes play any significant role in the reduced Cmax and total body exposure remain to be elucidated.

1. Chou, Chi-Chung, chingchun@nchu.edu.tw, Presenter
2. Wu, Chi-Ming, College of Veterinary Medicine,National Chung-Hsing University, Author
3. Lu, Yi-Ping, Biology Division, Veterinary Research Institute, Author
4. Rairat, Tirawat, Faculty of Fishery, Kasetsart University, Author
5. Liao, Ching-Chun, chingchun@nchu.edu.tw, Author

Barley as a production platform for oral vaccines in sustainable fish aquaculture

Fish health remains a major bottleneck in carp aquaculture. Beyond water quality, which can considerably weaken their immunity, carp are highly susceptible to viral, bacterial, fungal, and parasitic infections. Because these pathogens persist in pond systems, total eradication is unrealistic, and recent reductions in inflow water make it increasingly difficult to maintain optimal pond conditions. Alongside general preventive measures, species-specific vaccines could substantially improve fish survival. Yet no carp vaccine is currently registered in the European Union, even though many groups are pursuing its development.

An oral vaccine would be the best solution for practical use in farms, as its administration would not require excessive handling of the fish or special technical equipment. However, oral vaccines face a notable limitation due to insufficient stimulation of the complex gut-associated lymphoid tissue caused by factors such as vaccine degradation and poor absorption. Plant cells enable targeted storage of recombinant vaccines in specific cell organelles to ensure superior protection and contain natural compounds acting as adjuvants, which could lead to more efficient immune system activation.

Here we evaluate barley (Hordeum vulgare) as a production platform for an oral subunit vaccine against the envelope glycoprotein of spring viraemia of carp virus (SVCV). Lyophilised plant material was delivered to common carp (Cyprinus carpio) by intubation, and fish were sampled at 3, 7, 14, 21 and 28 days post-immunisation (dpi) to asses the induced immune responses on both cellular and molecular levels using a combination of flow cytometry and transcriptional analyses.

The results suggest a potent capacity of the plant material in inducing immune responses, manifested by an early influx of inflammatory myeloid cells to the intestinal tissue, accompanied by a progressive, 3—to 8-fold up-regulation of immunoglobulin genes across mucosal and systemic tissues. Collectively, these findings demonstrate that barley-based recombinant antigens can successfully prime carp immunity, highlighting the potential of plant-based vaccines for aquaculture.

1. KORYTAR, TOMAS, BIOLOGY CENTRE CAS, Presenter
2. Mičůchová, A., Czech Advanced Technology and Research Institute (CATRIN), Palacký University Olomouc, Author
3. Frébort, I., Czech Advanced Technology and Research Institute (CATRIN), Palacký University Olomouc, Author
4. Sebastian, A., Faculty of Fisheries and Protection of Waters, University of South Bohemia in České Budějovice, Author
5. Adamek, M., Fish Disease Research Unit, Institute of Parasitology, University of Veterinary Medicine Hannover, Author
6. Kyslík, J., Biology Centre CAS, Institute of Parasitology, Author
7. Piačková, V., 1Faculty of Fisheries and Protection of Waters, University of South Bohemia, Author

EVALUATING THE DIETARY INCLUSION OF A THYMOL-BASED BLEND TO MODULATE RESISTANCE AND SURVIVAL AGAINST Yersinia ruckeri IN RAINBOW TROUT

Yersinia ruckeri, the causative agent of enteric redmouth disease (ERM), is a common pathogen that causes substantial economic losses in salmonid aquaculture.

This study evaluated the efficacy a thymol-based blend – Prototype TCS – in reducing mortality on juvenile rainbow trout in following immersion challenge with Yersinia ruckeri. Two practical diets were manufactured to contain 2000 ppm of Prototype TCS and no additive (Control) and fed to 320 trouts divided in 16 tanks (n=8). After four weeks of feeding, the fish were challenged with Y. ruckeri. Fish fed diets containing Prototype TCS effectively reduced post-challenge mortality by 15.6% and hazard ratio by 29.3% (P<0.05 -Figure 1). Additionally, fish fed Prototype TCS had a significantly lower prevalence (P<0.05) of pathological signs in survivors, supporting enhanced disease robustness.

Our results demonstrated that including Prototype TCS in rainbow trout diets significantly increased resistance against ERM. This additive could therefore serve as promising alternatives to reduce antibiotic reliance and support more sustainable aquaculture practices.

1. SABBADIN ZANUZZO, FABIO, ONDA, Presenter
2. Liu, Angela, ONDA, Author
3. Poley, Jordan, ONDA, Author
4. Caruso, Fabrizio, VetAgro, Author
5. Leonardi, Geronimo, VetAgro, Author
6. Grilli, Ester, VetAgro, Author

Increasing the immunogenicity of a salmon louse labial gland protein in a DNA vaccine

Introduction:
Developing vaccines against ectoparasites presents significant challenges due to the limited exposure of these parasites to the host immune system. Effective immunization strategies must rely on immune effectors that impact the attachment site, hinder development, and/or alter the parasite’s behavior during feeding. Potential antigen for a vaccine against the ectoparasite salmon louse (Lepeophtheirus salmonis) is immunomodulatory proteins, which are deposited on the skin and modulate the immune response of Atlantic salmon. Such a vaccine would not directly target the lice, but would induce neutralizing antibodies against these immune-dampening proteins, thereby enabling the host to mount a more robust immune response towards the salmon louse. DNA vaccine technology is particularly suitable for this purpose, as such a vaccine must include several antigens. Given that DNA vaccines typically induce low antibody responses, additional measures were implemented to enhance their efficacy. Variations of synthetic hybrid constructs, combining a labial gland protein, a linker sequence, and flagellin from Moritella viscosa, were tested to evaluate the generation of specific antibodies against a labial gland protein.
Methodology:
Two common-garden in vivo vaccination experiments were conducted on Atlantic salmon parr. In both experiments, the fish were vaccinated with a 50 µl dose of different prototype vaccines, each containing 2.5 x 10¹² plasmid copies, administered intramuscularly. The vaccinated fish were maintained in freshwater at 12°C. Muscle, blood, and mucus samples were collected at 3 days, 1 week, 2 weeks, and 8 weeks post-vaccination and analyzed using RT-qPCR, histology, and ELISA. In the second experiment, different electroporation regimes were tested to optimize DNA vaccine administration.
Results and Conclusions:
Preliminary testing showed that DNA vaccination with unmodified labial gland proteins as antigens does not stimulate a strong immune response. Considerably higher antibody responses were observed in vaccine prototypes that included the fused labial gland protein sequence with flagellin and constructs that had an increased antigen size by linking the labial gland protein sequence together with multiple repeats. Histology revealed an influx of immune cells at the injection site 2 weeks post-vaccination. The prototype DNA vaccines were safe for Atlantic salmon parr, with no mortalities and good K-factors observed 8 weeks post-vaccination. Intramuscular injections combined with electroporation resulted in increased antibody responses against the labial gland protein. The combination of synthetic hybrid constructs and electroporation shows potential to increase DNA vaccine efficiency and antibody generation against a target antigen in Atlantic salmon.

1. Øvergård, Aina-Cathrine, University of Bergen, Presenter
2. Nelson, Patrick Alexander, University of Bergen, Author
3. Borchel, Andreas, University of Bergen, Author