The plasma metabolome of Atlantic salmon as studied by 1H NMR spectroscopy using standard operating procedures: effect of aquaculture location and growth stage.

INTRODUCTION: Metabolomics applications to the aquaculture research are increasing steadily. The use of standardized proton nuclear magnetic resonance (1H NMR) spectroscopy can provide the aquaculture industry with an unbiased, reproducible, and high-throughput screening tool, which can help to diagnose nutritional and disease-related metabolic disorders in farmed fish. OBJECTIVE: Standard operating procedures developed for analysing (human) plasma by 1H NMR were applied to fingerprint the metabolome in plasma samples collected from Atlantic salmon. The aim was to explore the metabolome of salmon plasma in relation to growth stage and sampling site. METHODS: A total of 72 salmon were collected from three aquaculture sites in Norway (Lat. 65, 67, and 70 °N) and over two sampling events (December 2017 and November 2018). Plasma drawn from each salmon was measured by 1H NMR and metabolites were quantified using the SigMa software. The NMR data was analysed by principal component analysis (PCA) and ANOVA-simultaneous component analysis (ASCA). RESULTS: Important metabolic differences were evidenced, with adult salmon having a much higher content of very low-density lipoproteins and cholesterol in their plasma, while smolts displayed significantly higher levels of propylene glycol. Overall, 24% of the metabolite variation was due to the growth stage, whereas 12% of the metabolite variation was related to the aquaculture site and practice (p < 0.001). CONCLUSION: This study provides a baseline investigation of the plasma metabolome of the Atlantic salmon and demonstrates how 1H NMR metabolomics can be used in future investigations for comparing aquaculture practices and their influence on the fish metabolome.

Zebrafish: Housing and husbandry recommendations.

This article provides recommendations for the care of laboratory zebrafish (Danio rerio) as part of the further implementation of Annex A to the European Convention on the protection of vertebrate animals used for experimental and other scientific purposes, EU Commission Recommendation 2007/526/EC and the fulfilment of Article 33 of EU Directive 2010/63, both concerning the housing and care of experimental animals. The recommendations provide guidance on best practices and ranges of husbandry parameters within which zebrafish welfare, as well as reproducibility of experimental procedures, are assured. Husbandry procedures found today in zebrafish facilities are numerous. While the vast majority of these practices are perfectly acceptable in terms of zebrafish physiology and welfare, the reproducibility of experimental results could be improved by further standardisation of husbandry procedures and exchange of husbandry information between laboratories. Standardisation protocols providing ranges of husbandry parameters are likely to be more successful and appropriate than the implementation of a set of fixed guidance values neglecting the empirically successful daily routines of many facilities and will better reflect the wide range of environmental parameters that characterise the natural habitats occupied by zebrafish. A joint working group on zebrafish housing and husbandry recommendations, with members of the European Society for Fish Models in Biology and Medicine (EUFishBioMed) and of the Federation of European Laboratory Animal Science Associations (FELASA) has been given a mandate to provide guidelines based on a FELASA list of parameters, 'Terms of Reference'.

Comparision of a serological potency assay for furunculosis vaccines (Aeromonas salmonicida subsp. salmonicida) to intraperitoneal challenge in Atlantic salmon (Salmo salar L.).

Batch potency testing of salmonid vaccines is mainly performed by in vivo challenge, which requires a lot of animals and causes severe pain. Due to the animal welfare concerns associated with in vivo immunization challenge tests, methods which could refine, reduce or replace (3Rs) these tests are needed. The aim of this study was to assess the use of serological assay (immunization & antibody estimation with an enzyme-linked immunosorbent assay (ELISA) for batch potency testing of oil adjuvanted, inactivated commercial furunculosis vaccines. In total ten vaccines were included in the study: two commercial multi-component vaccines and two experimental single-component furunculosis vaccines with 5% and 20% antigen content (relative to the commercial vaccine), from two manufacturers. In addition two experimental single component vaccines based on A-layer positive and A-layer negative Aeromonas salmonicida respectively were included. Challenge and blood sampling were conducted 9 weeks post vaccination. There was a correlation between antibody response against A. salmonicida as measured by ELISA and protection in i.p. challenge. This study shows that the ELISA assay can be used for testing different vaccine formulations and can potentially replace in vivo challenge tests for batch potency testing of furunculosis vaccines.

Antibody responses correlate with antigen dose and in vivo protection for oil-adjuvanted, experimental furunculosis (Aeromonas salmonicida subsp. salmonicida) vaccines in Atlantic salmon (Salmo salar L.) and can be used for batch potency testing of vaccines.

Salmon farming has increased dramatically over last thirty years and a key to the success is the introduction of protective vaccines. In Norway, almost 100% of all Atlantic salmon are vaccinated prior to sea transfer. This extensive use of vaccines demands use of a lot of resources in production and quality control of vaccines, and fish are now one of the most widely used laboratory animal species in Norway, since all batch testing today is performed by challenge experiments. With an increasing focus on the 3 R's (Replacement, Reduction and Refinement), new methods are needed. The aim of this study was to assess the use of different vaccine evaluation methods to identify furunculosis vaccines of different "potency", using ELISA as in vitro assay and intraperitoneal and cohabitation challenge as in vivo assays. Eleven vaccines with different antigen content (0, 2, 5, 10, 20, 40, 80, 100 and 200%) and different antigen qualities were included in the study. Challenge and blood sampling for the ELISA assay were conducted 9 weeks post vaccination. The results from this study indicated that there is a close correlation between the antigen dose in the vaccine and the antibody response against Aeromonas salmonicida as measured by ELISA. There is also a close correlation between the antibody response and protection for both i.p. and cohabitation challenge models. The ELISA method identified sub-potent batches better than currently used in vivo assay (i.p challenge) and seems to be the best method of performing a batch potency test of furunculosis vaccines particularly when taking the 3R's principles into account.

Preliminary experiments on use of zebrafish as a laboratory model for Giardia duodenalis infection.

Although Giardia duodenalis is considered a parasite of mammals, different genotypes have been identified as infecting several species of freshwater and marine fish in Australia. Establishment of G. duodenalis infection in common laboratory zebrafish (Danio rerio), could provide an excellent tool for a range of studies on Giardia. We conducted preliminary experiments to investigate this possibility. Zebrafish were inoculated with viable G. duodenalis cysts from two different Assemblages (A and D) using a modified oro-gastric tube. Direct microscopy and immunofluorescent antibody test were used to check for Giardia cysts/trophozoites in the intestine, and histology was performed on intestinal mucosa to evaluate possible pathological changes. Giardia cysts were successfully deposited in the zebrafish alimentary tract using a modified oro-gastric tube, and were maintained in the fish gut for at least 8 days. Although a single trophozoite was observed in one fish three days post-exposure, we were unable to demonstrate established, propagative infection under the conditions tested.

Impact of antibiotic treatments on the expression of the R plasmid tra genes and on the host innate immune activity during pRAS1 bearing Aeromonas hydrophila infection in zebrafish (Danio rerio).

BACKGROUND: The transfer of R plasmids between bacteria has been well studied under laboratory conditions and the transfer frequency has been found to vary between plasmids and under various physical conditions. For the first time, we here study the expression of the selected plasmid mobility genes traD, virB11 and virD4 in the 45 kb IncU plasmid, pRAS1, conferring resistance to tetracycline, trimethoprim and sulphonamide, using an in vivo zebrafish infection- treatment model. RESULTS: Three days after oral infection of adult zebrafish with Aeromonas hydrophila harboring pRAS1, elevated expression of pro-inflammatory cytokine (TNF α, IL-1ß and IL-8) and complement C3 genes in the intestine coincided with disease symptoms. Tetracycline, trimethoprim and an ineffective concentration of flumequine given 48 h prior to sampling, strongly increased expression of plasmid mobility genes, whereas an effective dosage of flumequine resulted in lower levels of mRNA copies of these genes relative to placebo treatment. Following effective treatment with flumequine, and ineffective treatments with a low concentration of flumequine, with trimethoprim or with sulphonamide, the intestinal expression of immune genes was strongly induced compared to placebo treated control fish. CONCLUSIONS: Treatment of zebrafish infected with an antibiotic resistant (TcR, TmR, SuR) A. hydrophila with ineffective concentrations of flumequine or the ineffective antimicrobials tetracycline and trimethoprim strongly induced expression of genes mediating conjugative transfer of the R-plasmid pRAS1. Simultaneously, there was a strong induction of selected inflammatory and immune response genes, which was again evident in fish subjected to ineffective treatment protocols. Our findings point to the essential role of therapeutic practices in escalation or control of antibiotic resistance transfer, and suggest that antibiotic substances, even in sub-inhibitory concentrations, may stimulate innate defenses against bacterial infections.

Development of an antibody ELISA for potency testing of furunculosis (Aeromonas salmonicida subsp salmonicida) vaccines in Atlantic salmon (Salmo salar L).

The study was conducted in Atlantic salmon to establish the initial and basic scientific documentation for an alternative batch potency test for salmon furuculosis vaccines. We assessed the antibody response development for Aeromonas salmonicida vaccines at different immunisation temperatures (3, 12 and 18 °C), by an enzyme-linked-immunosorbent assay (ELISA) 3, 6, 9 and 12 weeks post vaccination, and the correlation between antibody response and protection in cohabitation challenge experiments performed 6 and 12 weeks post vaccination. Fish immunised with a vaccine containing full antigen dose had a significant increase in antibody response after 252 day degrees and the measured values correlated well with protection after 500 day degrees. Fish vaccinated with a reduced antigen dose showed a significant lower antibody response than fish vaccinated with the full dose vaccine at all samplings, and showed a similar low relative percent survival (RPS) in the challenges. The results from this study indicate that an antibody ELISA can discriminate between vaccines of different antigen content and the method may replace challenge tests in batch potency testing of furunculosis vaccines in Atlantic salmon. An immunisation temperature of 12 °C and sampling after 6-9 weeks, seemed to be the most appropriate time for using antibody responses to confirm batch potency.

Three Rs Approaches in the Production and Quality Control of Fish Vaccines.

The workshop on Three Rs Approaches in the Production and Quality Control of Fish Vaccines aimed a) to identify animal tests currently stipulated for the production and quality control of fish vaccines and to highlight animal welfare concerns associated with these tests; b) to identify viable options to replace, reduce, and refine animal use for fish vaccine testing; and c) to discuss the way forward and set out how the Three Rs may be implemented without jeopardizing the quality of the vaccines. The workshop participants - experts from academia, regulatory authorities, a scientific animal welfare organization, and the fish vaccine industry - agreed that efforts should be undertaken to replace the vaccination-challenge batch potency testing with tests based on antigen quantification or antibody response tests. Regulatory requirements of questionable scientific value and relevance for the quality of fish vaccines, such as the re-testing of batches produced outside Europe, or the double-dose batch safety test, should be re-considered. As an immediate measure the design of the current animal tests should be evaluated and modified in the light of refinement and reduction, for example, the number of unprotected control fish in vaccination-challenge tests should be reduced to the minimum.

Detection of infectious pancreatic necrosis virus in subclinically infected Atlantic salmon by virus isolation in cell culture or real-time reverse transcription polymerase chain reaction: influence of sample preservation and storage.

Infectious pancreatic necrosis, an important problem of the salmon industry worldwide, is caused by Infectious pancreatic necrosis virus (IPNV). Fish surviving an IPNV infection become virus carriers, and the identification of infected fish is highly relevant to disease control. The target organ for IPNV diagnosis is the kidney, where the virus persists, usually with low virus loads. The current study documents a real-time reverse transcription polymerase chain reaction (real-time RT-PCR) assay that proved 100 times more sensitive than a conventional RT-PCR. Cell culture and real-time RT-PCR were compared for their ability to detect IPNV in carrier Atlantic salmon kidney samples after different preservation and storage procedures. Storage of whole tissue at -80°C for 1 month and storage of tissue homogenized in transport medium (TM) at +4°C for 1 week before investigation in cell cultures resulted in a marked reduction of virus infectivity. For detection by real-time RT-PCR, storage of whole tissue was suboptimal, whereas storage of tissue homogenized in TM did not affect virus detection. The results of the present study demonstrate that both cell culture and real-time RT-PCR are reliable tests for the detection of low amounts of IPNV in kidneys of carrier Atlantic salmon, and both methods are relatively robust against minor preservation and storage deviations, or both. Preservation of tissues in RNA stabilization solution seems only necessary when samples are to be shipped at ambient temperatures or when laboratory testing might be delayed. Independent of detection method, these results indicate that for long-term storage, samples are best kept at -80°C after homogenization in TM.

Stochastic modelling of direct costs of pancreas disease (PD) in Norwegian farmed Atlantic salmon (Salmo salar L.).

An economic model for estimating the direct costs of disease in industrial aquaculture was developed to include the following areas: biological losses, extraordinary costs, costs of treatment, costs of prevention and insurance pay-out. Direct costs of a pancreas disease (PD) outbreak in Norwegian farmed Atlantic salmon were estimated in the model, using probability distributions for the biological losses and expenditures associated with the disease. The biological effects of PD on mortality, growth, feed conversion and carcass quality and their correlations, together with costs of prevention were established using elicited data from an expert panel, and combined with basal losses in a control model. Extraordinary costs and costs associated with treatment were collected through a questionnaire sent to staff managing disease outbreaks. Norwegian national statistics for 2007 were used for prices and production costs in the model. Direct costs associated with a PD-outbreak in a site stocked with 500,000 smolts (vs. a similar site without the disease) were estimated to NOK (Norwegian kroner) 14.4 million (5% and 95% percentile: 10.5 and 17.8) (NOK=euro0.12 or $0.17 for 2007). Production was reduced to 70% (5% and 95% percentile: 57% and 81%) saleable biomass, and at an increased production cost of NOK 6.0 per kg (5% and 95% percentile: 3.5 and 8.7).

Sensitive and rapid detection of infectious pancreatic necrosis virus by reverse transcription loop mediated isothermal amplification.

A new molecular diagnostic assay was developed for rapid and sensitive diagnosis of infectious pancreatic necrosis virus (IPNV) by using a one step, one tube reverse transcription loop-mediated isothermal amplification (RT-LAMP). A set of six LAMP primers was designed to amplify the target RNA by incubation with Bst DNA polymerase plus reverse transcriptase and the reaction was optimised at 65 degrees C for 60 min. Three different methods for detection of the amplified product by naked eye gave identical results to gel electrophoresis, which was run for confirmation. Negative results obtained with RNA from four other fish viruses confirmed the specificity of the test. The IPNV-RT-LAMP assay demonstrated superior analytical sensitivity compared to conventional RT-PCR conducted according to published methods (1:10(12) dilution of RNA extracted from an IPNV-infected cell culture supernatant vs. 1:10(6) for the conventional RT-PCR). The feasibility of the RT-LAMP assay for detection of IPNV RNA in clinical specimen was authenticated using kidney tissue samples from experimentally IPNV-infected Atlantic salmon (Salmo salar) post-smolts. The results suggest that the RT-LAMP is a rapid and highly sensitive diagnostic assay for IPNV which lends itself well to use in aquaculture health management and disease control.

DNA vaccination can protect Cyprinus Carpio against spring viraemia of carp virus.

Several DNA constructs containing the spring viraemia of carp virus (SVCV) glycoprotein (G) gene were investigated for their ability to induce protection against SVCV following injection into myofibres. The constructs were pooled into four groups and co-injected with a plasmid encoding murine granulocyte-macrophage colony-stimulating factor. Group 1 contained one full-length and two truncated G constructs under the control of the cytomegalovirus (CMV) promoter. Group 2 contained full-length constructs with the CMV promoter, the simian virus 40 promoter and a muscle-specific promoter. Group 3 contained constructs in which the G-gene was fused with a second gene in order to improve secretion of the G-protein or to enhance destruction of transfected myocytes by T cells. Group 4 contained constructs with the CMV-Intron A promoter in plasmids with or without CpG motifs. A small-scale trial in goldfish showed that antibody responses in at least half the fish were induced by three injections of plasmids from Groups 1 and 3 whereas T-cell like responses with stimulation indices of above 3 were induced in at least half the fish by Groups 2 and 4. A single-dose of each plasmid mix was then used to protect carp in a large-scale trial. Following challenge with a heterologous strain of SVCV that killed 64% of fish, the strongest protection was observed in carp that received the full length G-gene expressed by two plasmids driven by the CMV-Intron A promoter (Group 4), with a relative percentage survival of 48% (p=0.00008).

MHC polymorphism and disease resistance in Atlantic salmon (Salmo salar); facing pathogens with single expressed major histocompatibility class I and class II loci.

Few studies have yet addressed the functional aspects of MHC molecules in fish. To lay the foundation for this, we evaluated the association between disease resistance and MHC class I and class II polymorphism in Atlantic salmon. Standardized disease challenge trials were performed on a semi-wild Atlantic salmon population with subsequent MHC typing and statistical analysis. The pathogens employed were infectious salmon anaemia virus (ISAV) causing infectious salmon anaemia and the Aeromonas salmonicida bacteria causing furunculosis. The material consisted of 1,182 Atlantic salmon from 33 families challenged with A. salmonicida and 1,031 Atlantic salmon from 25 families challenged with ISAV. We found highly significant associations between resistance towards infectious diseases caused by both pathogens and MH class I and class II polymorphism in Atlantic salmon. The observed associations were detected due to independently segregating MH class I and class II single loci, and inclusion of a large number of fish allowing an extensive statistical analysis.