Disease Of Atlantic Salmon Research Articles

Characterization of Tenacibaculum maritimum isolated from diseased salmonids farmed in Chile reveals high serological and genetic heterogeneity.

The diversity of Tenacibaculum maritimum in Chile remains poorly understood, particularly in terms of antigenic and genetic diversity. This information is crucial for the future development of a vaccine against tenacibaculosis and would increase understanding of this important fish pathogen. With this aim, the biochemical, antigenic, and genetic characteristics were analysed for 14 T. maritimum isolates, recovered from diseased Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss) farmed in Chile between 1998 and 2022. Biochemical analysis showed a homogeneity among all the Chilean T. maritimum isolates and all four other strains included for comparison purposes. Serological characterization using dot-blot assaying revealed antigenic heterogeneity with the use of unabsorbed antisera. The majority of isolates showed cross-reactions, identifying three main serological patterns. When the PCR-based serotyping scheme was performed, the existence of antigenic heterogeneity was confirmed. Four Atlantic salmon isolates were 4-0; and most isolates, including the rainbow trout isolate, were 3-1 (n = 9). A turbot (Scophthalmus maximus) isolate was 1-0. Using an existing Multilocus Sequence Typing system, two newly identified sequence types (ST193 and ST198) in the database were detected. ST193 encompassed nine isolates obtained from Atlantic salmon and rainbow trout, while ST198 regrouped four isolates, all retrieved from diseased Atlantic salmon in 2022. These findings highlight significant antigenic and genetic diversity among the Chilean isolates. This information is useful for epizootiology and the selection of suitable candidate strain(s) for vaccine development against tenacibaculosis caused by T. maritimum in Chilean salmon farming.

Transfer and bioaccumulation of chemical and biological contaminants in the marine polychaete Hediste diversicolor (OF müller 1776) when reared on salmon aquaculture sludge

Side streams from aquaculture production such as fish sludge poses ample opportunities for biological upcycling, as the sludge contains high amounts of nutrients, energy and valuable biochemicals, making it an ideal food for extractive species. Sludge has been proposed as a feed stock for polychaete production, which in turn can be utilized live in shrimp aquaculture or as an aquafeed ingredient. However, the biosafety of such value chains has not yet been addressed. We conducted an experiment exposing the polychaete Hediste diversicolor to aquaculture sludge spiked with four different fish pathogens (Mycobacterium salmoniphilum, Yersinia ruckeri, Infectious Pancreatic Necrosis (IPN) and Infectious Salmon Anaemia (ISA)) known to cause diseases in Atlantic salmon (Salmo salar L.). Moreover, we assessed whether heavy metals and other potentially hazardous elements present in fish sludge bioaccumulates in the polychaetes. Neither of the bacteria nor viruses could be detected in the polychaetes after 14 days of continuous exposure. Seven of the 15 elements we analysed showed bioaccumulation factors significantly below one, meaning biodilution, while the other eight did not differ from one, meaning no bioaccumulation. None of the elements showed a significant bioaccumulation. Further on, none of the heavy metals found in the polychaetes at the end of our experiment exceeded the EU regulatory maximum levels for fish feed ingredients. The current results suggest that a H. diversicolor can reared on aquaculture sludge, and aquaculture sludge may serve as feed stock for polychaete production without the product exceeding EU regulations for contaminants in animal feed.

A novel high-throughput qPCR chip for solving co-infections in RAS farmed rainbow trout

Recirculating aquaculture systems (RAS) have become more attractive due to reduced water consumption and effluent discharge. However, intensification of production increases the risk of introducing pathogens at farming sites. The emergence of uncultivable pathogens and RAS pathobiome diversity shifts the traditional disease paradigm from “one pathogen, one disease” to complex multiple-pathogen disease cases. Piscine orthoreovirus genotype 3 (PRV-3) is an excellent example, as it is capable of inducing anemia and heart pathology resembling heart and skeletal muscle inflammation under experimental conditions, and is associated with increased mortality in association with other pathogens in the field. The aim of this study was to develop a method for detection of multiple pathogens and putative pathogens, as co-infections are common in aquaculture. To do this, in the pilot study, we mapped the pathobiome of RAS-farmed rainbow trout (Oncorhynchus mykiss) (commercial RAS, farm A) using both standard diagnostic methods and metabarcording (16S rRNA) to investigate the gill microbiome. During this study, we observed infections with multiple pathogens, and detected two putative gill pathogens Candidatus Branchiomonas cysticola and Candidatus Piscichlamydia salmonis, both of which have been linked with complex gill disease in Atlantic salmon (Salmo salar). Based on the pilot study, we developed and tested a high throughput qPCR (HT-qPCR) chip targeting 22 viral and bacterial pathogens and putative pathogens, followed by a surveillance of a fish cohort in a commercial RAS farm during production (farm B). Co-infection with PRV-3 and Ca. B. cysticola combined with stress inducing management practices may explain the severe disease outbreak observed (37% mortality). The time course study sets the base for a future screening scheme for disease prediction and addresses limitations of the method when testing environmental DNA/RNA.

Persistent immune responses in the heart determine the outcome of cardiomyopathy syndrome in Atlantic salmon (Salmo salar)

Cardiomyopathy syndrome (CMS) caused by piscine myocarditis virus (PMCV) is a severe cardiac disease in Atlantic salmon (Salmo salar) and one of the leading causes of morbidity and mortality in the Norwegian aquaculture industry. Previous research suggest a variation in individual susceptibility to develop severe disease, however the role of the immune response in determining individual outcome of CMS is poorly understood particularly in cases where fish are also challenged by stress. The present study's aim was therefore to characterize cardiac transcriptional responses to PMCV infection in Atlantic salmon responding to infection under stressful conditions with a high versus low degree of histopathological damage.The study was performed as a large-scale controlled experiment of Atlantic salmon smolts from pre-challenge to 12 weeks post infection (wpi) with PMCV, during which fish were exposed to intermittent stressors. RNA sequencing (RNAseq) was used to compare the heart transcriptome of high responders (HR) with atrium histopathology score ‘4’ and low responders (LR) with score ‘0.5’ at 12 wpi. A high-throughput quantitative PCR (qPCR) analysis was used to compare immune gene transcription between individuals sampled at 6, 9 and 12 wpi. Based on RNAseq and qPCR results, RNAscope in situ hybridization (ISH) was performed for visualization of IFN-γ - and IFNb producing immune cells in affected heart tissue.Compared to LR, the transcription of 1592 genes was increased in HR at 12 wpi. Of these genes, around.40 % were immune-related, including various chemokines, key antiviral response molecules, and genes.associated with a Th1 pro-inflammatory immune response. Further, the qPCR analysis confirmed.increased immune gene transcription in HR at both 9 and 12 wpi, despite a decrease in PMCV.transcription between these time points. Interestingly, increased IFNb transcription in HR suggests the.presence of high-quantity IFN secreting cells in the hearts of these individuals. Indeed, RNAscope.confirmed the presence of IFN-γ and IFNb-positive cells in the heart ventricle of HR but not LR.To conclude, our data indicate that in severe outcomes of PMCV infection various chemokines attract leucocytes to the salmon heart, including IFN-γ and IFNb-secreting cells, and that these cells play important roles in maintaining persistent antiviral responses and a sustained host immunopathology despite decreasing heart viral transcription.

Effectiveness of functional ingredients to enhance gill disease in Atlantic salmon (Salmo salar, L.).

The development and application of functional feed ingredients represents a great opportunity to advance fish growth and health, boost the immune system, and induce physiological benefits beyond those provided by traditional feeds. In the present study, we looked at the feasibility of in vitro methods for screening the qualities of functional feed ingredients using the fish cell line RTgill-W1, which has never been used in fish nutrition, and the culture of Paramoeba perurans. Five functional feed ingredients (arginine, β-glucan, vitamin C, and two phytogenic feed additives) were selected to investigate their effects on cell viability and reactive oxygen species production. Three of the selected ingredients (arginine and two phytogenic feed additives) were additionally tested to assess their potential amoebicidal activity. As these functional ingredients are the core of a commercially available feed (Protec Gill, Skretting AS), their beneficial effects were further assessed in a field trial in fish affected by complex gill disease. Here, the analyzed parameters included the evaluation of macroscopic and histopathological gill conditions, pathogen detections, and analyses of plasma parameters. RTgill-W1 cell line assays were a good tool for screening functional ingredients and provided information about the optimal ingredient concentration ranges, which can be helpful for adjusting the concentrations in future feed diets. Through the culture of P. perurans, the tested ingredients showed a clear amoebicidal activity, suggesting that their inclusions in dietary supplements could be a viable way to prevent microbial infections. A three-week period of feeding Protec Gill slowed the disease progression, by reducing the pathogen load and significantly improving gill tissue conditions, as revealed by histological evaluation. The use of diets containing selected functional ingredients may be a feasible strategy for preventing or mitigating the increasingly common gill diseases, particularly in cases of complex gill disease, as documented in this study.

Comparative Genomic Analysis of a Novel Vibrio sp. Isolated from an Ulcer Disease Event in Atlantic Salmon (Salmo salar).

Ulcer diseases are a recalcitrant issue at Atlantic salmon (Salmo salar) aquaculture cage-sites across the North Atlantic region. Classical ulcerative outbreaks (also called winter ulcer disease) refer to a skin infection caused by Moritella viscosa. However, several bacterial species are frequently isolated from ulcer disease events, and it is unclear if other undescribed pathogens are implicated in ulcer disease in Atlantic salmon. Although different polyvalent vaccines are used against M. viscosa, ulcerative outbreaks are continuously reported in Atlantic salmon in Canada. This study analyzed the phenotypical and genomic characteristics of Vibrio sp. J383 isolated from internal organs of vaccinated farmed Atlantic salmon displaying clinical signs of ulcer disease. Infection assays conducted on vaccinated farmed Atlantic salmon and revealed that Vibrio sp. J383 causes a low level of mortalities when administered intracelomic at doses ranging from 107-108 CFU/dose. Vibrio sp. J383 persisted in the blood of infected fish for at least 8 weeks at 10 and 12 °C. Clinical signs of this disease were greatest 12 °C, but no mortality and bacteremia were observed at 16 °C. The Vibrio sp. J383 genome (5,902,734 bp) has two chromosomes of 3,633,265 bp and 2,068,312 bp, respectively, and one large plasmid of 201,166 bp. Phylogenetic and comparative analyses indicated that Vibrio sp. J383 is related to V. splendidus, with 93% identity. Furthermore, the phenotypic analysis showed that there were significant differences between Vibrio sp. J383 and other Vibrio spp, suggesting J383 is a novel Vibrio species adapted to cold temperatures.

Co-occurrence of heterogeneous Flavobacterium psychrophilum isolates within the same Chilean farm and during the same infectious outbreak.

Flavobacterium psychrophilum is a pathogenic bacterium affecting Chilean salmonid farms. High antigenic and genetic diversity exists among Chilean F. psychrophilum isolates, but the distribution thereof among farms is poorly understood. These epidemiological data are key for developing isolate-specific vaccines. The present study isolated F. psychrophilum in diseased Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss) from five freshwater farms between 2018 and 2019. Each farm only raised one salmonid species and was geographically separated from and did not share culturing water with the other farms. Antigenic and genetic analyses were conducted to shed light on the possibility of isolates coexisting within the same farm during outbreaks. A total of 68 Chilean F. psychrophilum isolates were recovered from skin lesions, gills, fins, kidney and spleen of moribund and live fish. Among the 68 Chilean isolates, mPCR-serotyping indicated three major serotypes (i.e. 23.5% type 0; 47.1% type 2; and 26.5% type 4) and, to a lesser degree, serotype 1 (2.9%). Sixteen antigenic groups were detected by slide agglutination. Genetic characterizations by 16S rRNA alleles identified 71% of the isolates as the virulent genogroup CSF259-93 allele. A predominant serotype was associated with each farm, with types 0 and 4 related to Atlantic salmon and types 1 and 2 to rainbow trout. Notwithstanding, several antigenic groups coexisted within some farms. Likewise, the experimental intramuscular challenges (n = 20) demonstrated that the type-2 isolates from rainbow trout were the most pathogenic among isolates recovered from infectious outbreaks in Atlantic salmon, especially as compared to those from types-0 and -4. These results allow us to suggest that prevention measures, specifically vaccines, should be developed according to dominant isolates and with specificity to each farm, that is the use of autogenous or site-specific vaccines.

Moritella viscosa early infection and transcriptional responses of intraperitoneal vaccinated and unvaccinated Atlantic salmon

Skin is the first line of defence against waterborne pathogens, such as Moritella viscosa, which is the causative agent of winter ulcer disease in Atlantic salmon (Salmo salar L.). The present study revealed that vaccination protects against both M. viscosa-induced mortality and skin ulceration, but protection varied according to the strain used in the vaccine formulation. Examination of skin tissue 4 days post M. viscosa challenge indicated that M. viscosa initiated infection by colonising the scale surface. Sequencing the transcripts of the variable region of the IgM heavy chain did not detect effect of vaccination or M. viscosa challenge on antibody repertoire and B cell trafficking from the lymphatic organs to skin. Skin layer-specific responses were examined with 44 K oligonucleotide microarray. Skin responded to vaccination and M. viscosa challenge by increasing transcription of structural genes, stimulating metabolism, and regulation of developmental processes. Immune responses to dead (vaccine) and live M. viscosa were very similar and no pathogen-specific changes were found. Combined use of skin transcriptional profiles and image analysis revealed differences in the infected skin layers between the study groups. M. viscosa was likely retained in the epidermis of vaccinated salmon, whereas the dermis was colonised in unvaccinated fish. Furthermore, decreased expression of lymphocyte-specific and antiviral genes in the dermis indicated possible evasion of immune-related cells from skin in salmon challenged with M. viscosa. Our data also indicated that intraperitoneal vaccination may prime the humoral innate response, which enables a rapid response in the dermis layer of skin during M. viscosa invasion.

Prospect of Hydroxyl Radical Exposure during Seawater Bathing to Treat Amoebic Gill Disease in Atlantic Salmon

This study aims to undertake hydroxyl (.OH) radical-based preliminary investigations with a view to utilising seawater as a viable alternative to freshwater for the treatment of amoebic gill disease (AGD) in aquaculture industries. The study presents in vitro viability studies of clonal amoebae species to examine the effects of .OH radicals on both parasites and hosts. The study also assesses the toxicity to Chinook salmon cell lines (CHSE-214) in freshwater and 35 ppt seawater via continuous dosing of 35 mM .OH radicals and hydrogen peroxide (H2O2) for 1.5 to 4 hr, separately at 18°C and 15°C. Comparatively high viability of CHSE-214 (60% in .OH and 50% in H2O2) for a prolonged treatment of up to 4 hr in seawater at 15°C indicates suitability of low seawater temperature while using either .OH or H2O2 during bathing. The viability of CHSE-214 remained relatively stable in seawater (55%–60% in .OH and 50%–60% in H2O2), at both temperatures of 18°C and 15°C. However, at 15°C, a drastic reduction of viability of CHSE-214 in freshwater (from 80% to 48% in .OH and from 70% to 58% in H2O2) has indicated high variations in toxicity levels in freshwater at low temperature. Using DNA staining agents in flow cytometry, the in vitro viability study results in >22.5% mortality of clonal Neoparamoeba perurans (NP), an AGD causative agent, in 35 ppt seawater containing 35 mM .OH radicals via one-off dosing for 1 hour at 15°C. In addition, fast radical consumption is more pronounced in the case of .OH radicals as compared to H2O2 in both freshwater and seawater due to extreme reactivity of the former. Hence, this study suggests that .OH radicals are detrimental to the viability of NP in seawater, and thereby, establishes grounds for further in vivo investigations of using seawater supplemented with continuous dosing of .OH radicals for Atlantic salmon bathing as a treatment of AGD.

Expression Analysis of Moritella viscosa-Challenged Atlantic Salmon Identifies Disease-Responding Genes, MicroRNAs and Their Predicted Target Genes and Pathways.

Moritella viscosa is a bacterial pathogen causing winter-ulcer disease in Atlantic salmon. The lesions on affected fish lead to increased mortality, decreased fish welfare, and inferior meat quality in farmed salmon. MicroRNAs (miRNAs) are small non-coding RNAs involved in post-transcriptional regulation by guiding the miRNA-induced silencing complex to specific mRNA transcripts (target genes). The goal of this study was to identify miRNAs responding to Moritella viscosa in salmon by investigating miRNA expression in the head-kidney and the muscle/skin from lesion sites caused by the pathogen. Protein coding gene expression was investigated by microarray analysis in the same materials. Seventeen differentially expressed guide-miRNAs (gDE-miRNAs) were identified in the head-kidney, and thirty-nine in lesion sites, while the microarray analysis reproduced the differential expression signature of several thousand genes known as infection-responsive. In silico target prediction and enrichment analysis suggested that the gDE-miRNAs were predicted to target genes involved in immune responses, hemostasis, angiogenesis, stress responses, metabolism, cell growth, and apoptosis. The majority of the conserved gDE-miRNAs (e.g., miR-125, miR-132, miR-146, miR-152, miR-155, miR-223 and miR-2188) are known as infection-responsive in other vertebrates. Collectively, the findings indicate that gDE-miRNAs are important post-transcriptional gene regulators of the host response to bacterial infection.

Effects of a DNA and multivalent oil-adjuvanted vaccines against pancreas disease in Atlantic salmon (Salmo salar) challenged with salmonid alphavirus subtype 3

Salmonid alphavirus (SAV) causes pancreas disease (PD) in Atlantic salmon (Salmo salar). In seawater-farmed salmonids in the southern part of Norway SAV subtype 3 (SAV3) is dominating. PD continues to cause significant economic and fish health concerns in this region despite years of extensive use of oil-adjuvanted vaccines (OAVs) containing inactivated whole virus (IWV) antigens. In the current study, three commercially available PD vaccines were tested. Group A got a DNA vaccine (DNAV) injected intramuscularly (i.m.) plus an OAV without a PD component injected intraperitoneally (i.p.). Groups B and C got different OAV IWV vaccines injected i.p., respectively. The control group was i.p. injected with saline. Approximately 12 weeks after vaccination, the post smolt groups were challenged in seawater with SAV3 by cohabitation. Samples were collected pre-challenge, and at 19, 54 and 83 days post-challenge (dpc). There were no differences in growth or visible intraperitoneal side effects between the immunized groups prior to challenge. Fish in group A had significantly higher SAV3 neutralizing antibody titers than the other groups pre-challenge and significantly lower SAV3 viremia levels than the control group at 19 dpc. Fish in group A had significantly more weight gain than the other groups measured at 54 and 83 dpc. Prevalence and severity of heart necrosis at 19 dpc and loss of exocrine pancreas tissue at 54 and 83 dpc were significantly lower in groups A and B compared to group C and controls. The cumulative mortality in the control group during the challenge period was 10.5%. Group A experienced the lowest mortality (6.4%) albeit not statistically different from the controls. The results suggest that DNAV may reduce the clinical and economic impact of PD by improved protection against SAV3-induced changes in pancreas tissue and growth impairment.

Investigating the impacts of H2O2 treatment on gills of healthy Atlantic salmon reveals potential changes to mucus production with implications on immune activity

Current treatment strategies for relevant infectious diseases in Atlantic salmon (Salmo salar L.) include the use of low salinity or freshwater bathing. However, often availability is restricted, and hydrogen peroxide (H2O2) is used as an alternative. The potential impacts of H2O2 on fish mucosal tissues, especially the gills therefore need to be considered. In this study the mucosal and immunological effects of H2O2 treatment on the gills of healthy Atlantic salmon were examined by gene expression (qPCR) and immunohistochemistry (IHC) investigating T-cell, B-cell, and mucin activity. Healthy fish were treated with H2O2 and sampled at different times: 4 h, 24 h and 14 days post-H2O2 treatment (dpt) (total n = 18) to investigate the effect of holding time and H2O2 treatment. Treatment with H2O2 resulted in up-regulation of markers for T-cell activity and anti-inflammatory response and down-regulation of mucin expression in the gills at 14 dpt compared to fish sampled prior to treatment (0h; n = 5 fish). These findings were supported by IHC analysis, which despite being highly variable between samples, showed an increase in the number of CD3+ T cells at 14 dpt in 50% of treated fish compared to pre-treatment fish. The results from this study suggest that H2O2 treatment does not immune compromise healthy Atlantic salmon after 14 dpt (i.e., post-recovery) but modulates gill immune activity and disrupts the mucus covering of the gills. However, further studies are required to determine whether the effects observed are related to H2O2 treatment in isolation or other variables such as holding time or environmental factors.

Novel DNA‐based in situ hybridization method to detect Desmozoon lepeophtherii in Atlantic salmon tissues

The microsporidian Desmozoon lepeophtherii Freeman and Sommerville, 2009 is considered significant in the pathogenesis of gill disease in Atlantic salmon (Salmo salar Linnaeus, 1758). Due to the difficulty in detecting D.lepeophtherii in tissue sections, infections are normally diagnosed by molecular methods, routine haematoxylin and eosin (H&E) stained gill tissue sections and the use of other histochemical stains and labels to confirm the presence of spores. An in situ hybridization (ISH) protocol specific for D.lepeophtherii was developed using DIG-labelled oligonucleotide probes. Diseased Atlantic salmon gills were analysed by ISH, calcofluor white (CW) and H&E. All methods showed high levels of specificity (100%) in their ability to detect D.lepeophtherii, but the sensitivity was higher with ISH (92%), compared with CW (64%) and the presence of microvesicles on H&E stained sections (52%). High levels of D.lepeophtherii spores were significantly associated (p<.05) with the development of D.lepeophtherii-associated pathology in the gills, with Ct values below 19 and over 100 microsporidia/10 mm2 of gill tissue (from the ISH counts) seemingly necessary for the development of microvesicles. The ISH method has the advantage over other histological techniques in that it allows all life stages of the microsporidian to be detected in infected salmon gill tissue sections.

Pasteurella skyensis in Atlantic salmon (Salmo salar L.) in Western Norway

Pasteurellosis has been an emerging disease in Atlantic salmon (Salmo salar L) in Western Norway in recent years. This paper presents the first case study from Atlantic salmon in Western Norway where the cause of mortality and morbidity was associated with Pasteurella skyensis.

Multi-agent in situ hybridization confirms Ca. Branchiomonas cysticola as a major contributor in complex gill disease in Atlantic salmon

Gill diseases may cause high mortalities in farmed Atlantic salmon. In seawater reared fish co-infections involving the epitheliocystis associated bacterium Ca. Branchiomonas cysticola, the microsporidian Desmozoon lepeophtherii, the causative agent of amoebic gill disease Paramoeba perurans and salmon gill poxvirus are common and histopathological lesions may be complex. Here, we report detection of these agents utilising multiplex real-time PCR and link the presence of agents to histopathologically visible gill lesions by in situ hybridisation (ISH) utilising RNAscope®. We show that Ca. Branchiomonas cysticola infections may remain undetected if diagnostic investigations are restricted to histopathology alone. Further, positive in situ labelling of Ca. Branchiomonas cysticola was observed within epitheliocysts, but also in small foci within areas of inflammation and necrosis in which histologically detectable epitheliocysts were not visible. In situ labelling of D. lepeophtherii corresponded well with tissue distribution patterns previously associated with this microsporidian. Salmon gill poxvirus was associated with apoptotic gill epithelial cells, while Ca. Piscichlamydia salmonis could not be associated with pathological changes. The multiplex real-time PCRs utilised were rapid and sensitive diagnostic tools and the results corresponded well with ISH. This study shows that the agents involved in complex gill disease can be linked to lesions using ISH and suggests that Ca. B. cysticola plays a crucial role in the development of gill disease in the farming of salmon in Norway.

Development of Aeromonas salmonicida subsp. masoucida vaccine in turbot and evaluation of protection efficacy under field conditions

Aeromonas salmonicida is a global fish pathogen that can infect a series of fish species. Vaccination by commercial vaccine has been proved to be an effective method to control this disease in Atlantic salmon. However, commercial vaccines used to protect against A. salmonicida subsp. salmonicida infection are not necessarily effective against infections by other subspecies. Given increasing number of infections by A. salmonicida subsp. masoucida in turbot (Scophthalmus maximus) farming in China, we developed an A. salmonicida subsp. masoucida inactivated vaccine for turbot in the present research. Using a previously isolated pathogenic strain RZ6S-1, a water-based vaccine and an oil-based Marcol 52 emulsified vaccine were developed, and first evaluated under laboratory conditions. Results showed that the relative percent of survival (RPS) and specific serum antibody titers of the fish vaccinated by the emulsified vaccine were significantly higher than that of those vaccinated by the water-based vaccine at 4 weeks post vaccination. Under field conditions, turbot were protected for 12 weeks and 24 weeks post vaccination by emulsified vaccine, with the challenged RPS reaching 84.66 ± 6.39% and 84.54 ± 5.93%, respectively. The specific antibody titers against A. salmonicida subsp. masoucida in the vaccinated fish could even be detected at 24 weeks post vaccination. In comparison, cumulative mortality of the control fish (12.96%) was higher than that of the vaccinated fish (2.48%), with more fish died of natural infection by A. salmonicida (221 vs. 17). Cutaneous nodules, possibly caused by chronic infection by A. salmonicida, were found in 23 of 30 randomly selected fish in the control pond at 24 weeks post vaccination, in contrast to only 1 of 30 in the vaccinated pond. More infections caused by Edwardsiella piscicida and scuticociliate were also observed in the control pond, indicating that the cutaneous nodules may facilitate the infection by other pathogens and parasites. Additionally, the fish weight in the vaccinated pond was significantly higher than that in the control pond, both at 12 weeks and 24 weeks post vaccination. These results indicated that the A. salmonicida emulsified vaccine can protect turbot for at least 24 weeks without severe side-effects on the fish growth under field conditions, and can also protect fish from chronic infection, which in turn reduces the infection probability by other pathogens or parasites. In conclusion, the present study indicates that the developed vaccine protects turbot from A. salmonicida subsp. masoucida infection under farming conditions.

Application of Quantitative-PCR to Monitor Netpen Sites in British Columbia (Canada) for Tenacibaculum Species.

Tenacibaculum are frequently detected from fish with tenacibaculosis at aquaculture sites; however, information on the ecology of these bacteria is sparse. Quantitative-PCR assays were used to detect T. maritimum and T. dicentrarchi at commercial Atlantic salmon (Salmo salar) netpen sites throughout several tenacibaculosis outbreaks. T. dicentrarchi and T. maritimum were identified in live fish, dead fish, other organisms associated with netpens, water samples and on inanimate substrates, which indicates a ubiquitous distribution around stocked netpen sites. Before an outbreak, T. dicentrarchi was found throughout the environment and from fish, and T. maritimum was infrequently identified. During an outbreak, increases in the bacterial load in were recorded and no differences were recorded after an outbreak supporting the observed recrudescence of mouthrot. More bacteria were recorded in the summer months, with more mortality events and antibiotic treatments, indicating that seasonality may influence tenacibaculosis; however, outbreaks occurred in both seasons. Relationships were identified between fish mortalities and antimicrobial use to water quality parameters (temperature, salinity, dissolved oxygen) (p < 0.05), but with low R2 values (<0.25), other variables are also involved. Furthermore, Tenacibaculum species appear to have a ubiquitous spatial and temporal distribution around stocked netpen sites, and with the potential to induce disease in Atlantic salmon, continued research is needed.

Experimental exposure to low concentrations of Neoparamoeba perurans induces amoebic gill disease in Atlantic salmon.

Amoebic gill disease (AGD) is a significant issue in Atlantic salmon mariculture. Research on the development of treatments or vaccines uses experimental challenges where salmon is exposed to amoebae concentrations ranging from 500 to 5,000/L. However, the water concentrations of N.perurans on affected salmon farms are much lower. The lowest concentration of N.perurans previously reported to cause AGD was 10/L. Here, we report that concentrations as low as 0.1/L of N.perurans can cause AGD. We propose that concentrations of N.perurans that reflect those measured on salmon farms should be used for future experimental challenges.

Evaluation of sodium percarbonate as a bath treatment for amoebic gill disease in Atlantic salmon

Amoebic gill disease (AGD), caused by Neoparamoeba perurans, is a major health challenge for Atlantic salmon aquaculture globally. While freshwater bathing for 2 hr is effective in reducing infection severity, there is need for more rapid and lower cost alternatives. To this end, a combination of sodium percarbonate (SPC) in freshwater was examined for its treatment efficacy. Initial in vitro studies showed a reduction in amoeba viability when exposed for 30 min to freshwater containing >500 mg/L SPC. Subsequently, AGD-affected salmon were bathed for 30 min in 16°C freshwater containing 100, 500 or 1,000 mg/L SPC, or for 2 hr in 16°C freshwater to mimic industry practice. Treatment at the highest SPC concentration caused extensive gill damage and substantial mortality. Neither occurred to a significant extent at lower SPC concentrations. Gill pathology of surviving fish 10 days post-treatment (dpt) was comparable to or more severe than pre-treatment, and significantly (p < .001) more severe than in 2 hr freshwater bathed fish. N. perurans DNA was confirmed by qPCR in all treatment groups at 10 dpt. The data indicate that a 30-min exposure to SPC in freshwater is not a suitable alternative to existing freshwater treatment of AGD.

Data-Driven Network Modeling as a Framework to Evaluate the Transmission of Piscine Myocarditis Virus (PMCV) in the Irish Farmed Atlantic Salmon Population and the Impact of Different Mitigation Measures

Cardiomyopathy syndrome (CMS) is a severe cardiac disease of Atlantic salmon caused by the piscine myocarditis virus (PMCV), which was first reported in Ireland in 2012. In this paper, we describe the use of data-driven network modeling as a framework to evaluate the transmission of PMCV in the Irish farmed Atlantic salmon population and the impact of different mitigation measures. Input data included live fish movement data from 2009 to 2017, population dynamics events and the spatial location of the farms. With these inputs, we fitted a network-based stochastic infection spread model. After assumed initial introduction of the agent in 2009, our results indicate that it took 5 years to reach a between-farm prevalence of 100% in late 2014, with older fish being most affected. Local spread accounted for only a small proportion of new infections, being more important for sustained infection in a given area. Spread via movement of subclinically infected fish was most important for explaining the observed countrywide spread of the agent. Of the targeted intervention strategies evaluated, the most effective were those that target those fish farms in Ireland that can be considered the most connected, based on the number of farm-to-farm linkages in a specific time period through outward fish movements. The application of these interventions in a proactive way (before the first reported outbreak of the disease in 2012), assuming an active testing of fish consignments to and from the top 8 ranked farms in terms of outward fish movement, would have yielded the most protection for the Irish salmon farming industry. Using this approach, the between-farm PMCV prevalence never exceeded 20% throughout the simulation time (as opposed to the simulated 100% when no interventions are applied). We argue that the Irish salmon farming industry would benefit from this approach in the future, as it would help in early detection and prevention of the spread of viral agents currently exotic to the country.