Anatomical distribution of scavenger endothelial cells in bony fishes (Osteichthyes).

The scavenger endothelial cells (SECs) of vertebrates are an important class of endocytic cells responsible for clearance of foreign and physiological waste macromolecules, partitioning in the immune system, functioning as a cellular powerplant by producing high energy metabolites like lactate and acetate. All animal phyla possess SECs, but the tissue localization of SECs has only been investigated in a limited number of species. By using a specific ligand for scavenger receptors (formalin treated bovine serum albumin), the study revealed that in all tetrapod species (amphibia, reptiles, birds and mammals) the SECs were found lining the sinusoids of the liver. No SECs were found in the liver of any of the bony fishes (Osteichthyes) investigated. Interestingly, we found the SECs not only to be located in the heart of marine species but also in some freshwater species such as Lota lota, Percichthys trucha and Perca fluviatilis. In some fish species, the SECs were found both in the heart and/or kidney in a number of marine and freshwater fishes, whereas in some marine, diadromous and freshwater fishes the SECs were confined only to the kidney tissue. However, from these results it can be suggested that there is neither a clear phylogenetic trend when it came to anatomical localization of SECs nor any pattern in terms of habitat (salinity preferences).

Inflammatory responses in Atlantic lumpfish (Cyclopterus lumpus L.) after intraperitoneal injection of a vaccine against Aeromonas salmonicida and Vibrio salmonicida at different water temperatures.

Studying inflammatory responses induced by vaccination can contribute to a more detailed understanding of underlying immune mechanisms in lumpfish (Cyclopterus lumpus). Tissue samples from lumpfish intraperitoneally immunized with a divalent oil-adjuvanted vaccine (Aeromonas salmonicida and Vibrio salmonicida) at water temperatures of 5, 10, and 15°C were collected at 630 day degrees and 18 weeks post injection. The relative amount of secretory and membrane-bound immunoglobulin M (IgM) gene transcripts in the head kidney was determined by qPCR. Vaccine-induced inflammatory lesions were assessed on histological sections of abdominal pancreatic/intestinal tissue from vaccinated fish in all three temperature groups. Inflammatory cells forming dense aggregations in lesions showed proliferative activity, many of which were identified as eosinophilic-granulocyte-like cells. IgM+ cells were scattered in inflammatory tissue dominated by connective tissue, showing no difference in numbers between lesions from fish vaccinated at 5, 10, and 15°C. Relative gene expression analysis of secretory and membrane-bound IgM revealed low overall expression in the head kidney of vaccinated fish at both 630 day-degrees and 18 weeks post injection. The results of this study indicate that the vaccine stimulated prolonged local inflammatory responses at the injection site, which were not influenced by temperature.

CRISPR/Cas9-Mediated Gene Editing in Salmonids Cells and Efficient Establishment of Edited Clonal Cell Lines.

Finfish production has seen over three-fold increase in the past 30 years (1990-2020), and Atlantic salmon (A. salmon; salmo salar) accounted for approximately 32.6% of the total marine and coastal aquaculture of all finfish species in the year 2020, making it one of the most profitable farmed fish species globally. This growth in production is, however, threatened by a number of problems which can be solved using the CRISPR/Cas technology. In vitro applications of CRISPR/Cas using cell lines can complement its in vivo applications, but salmonids-derived cell lines are difficult to gene edit because they grow slowly, are difficult to transfect and isolate single clones of gene-edited cells. While clonal isolation of the gene-edited Chinook salmon cell line (CHSE-214) has successfully been performed, there is no report of successful clonal isolation of the gene-edited A. salmon ASK-1 and SHK-1cell lines. In the current study, two gene loci-cr2 and mmp9 of A. salmon-were efficiently edited using the ribonucleoprotein (RNP) and plasmid CRISPR/Cas9 strategies. Edited cells were enriched using flow cytometer-activated cell sorting (FACS), followed by clonal isolation and expansion of edited cells. The study both confirms the recent report of the highly efficient editing of these widely used model cell lines, as well as extends the frontline in the single-cell cloning of gene-edited salmonids cells. The report also highlights the pitfalls and future directions in the application of CRISPR/Cas9 in these cells.

Fluorescence fluctuation-based super-resolution microscopy using multimodal waveguided illumination.

Photonic chip-based total internal reflection fluorescence microscopy (c-TIRFM) is an emerging technology enabling a large TIRF excitation area decoupled from the detection objective. Additionally, due to the inherent multimodal nature of wide waveguides, it is a convenient platform for introducing temporal fluctuations in the illumination pattern. The fluorescence fluctuation-based nanoscopy technique multiple signal classification algorithm (MUSICAL) does not assume stochastic independence of the emitter emission and can therefore exploit fluctuations arising from other sources, as such multimodal illumination patterns. In this work, we demonstrate and verify the utilization of fluctuations in the illumination for super-resolution imaging using MUSICAL on actin in salmon keratocytes. The resolution improvement was measured to be 2.2-3.6-fold compared to the corresponding conventional images.

Scavenger endothelial cells of fish, a review.

The definition of scavenger endothelial cells (SEC) is exclusively based on functional and structural characteristics. The following characteristics are common hallmarks for the vertebrate SEC: (a) All vertebrates examined are furnished with a population of special SEC that plays a role in the catabolism of physiologic and non-physiologic soluble waste macromolecules. (b) From the ligands that are endocytosed, SEC in all seven vertebrate classes appear to express the collagen α-chain receptor and the scavenger receptors. In addition, the hyaluronan and the mannose receptors are present on SEC of mammalia (several species) and osteichthyes (e.g., salmon and cod). It is likely that all four receptor types are present in all vertebrate classes. (c) Like liver endothelial cells (LEC) in mammals, SEC in all vertebrate classes are geared to endocytosis of soluble macromolecules, but phagocytic uptake of particles is taken care of mainly by macrophages. (d) The most primitive vertebrates (hagfish, lamprey and ray) carry their SEC in gill vessels, whereas phylogenetically younger fishes (salmon, carp, cod and plaice) carry their SEC in either kidney or heart and in all terrestrial vertebrates-SEC are found exclusively in the liver. (e) SEC of all vertebrates are localized in blood sinusoids or trabeculae that carry large amounts of slowly flowing and O2 poor blood. (f) SEC differs functionally and structurally from what is normally associated with "conventional vascular endothelium."

Protection of Teleost Fish against Infectious Diseases through Oral Administration of Vaccines: Update 2021.

Immersion and intraperitoneal injection are the two most common methods used for the vaccination of fish. Because both methods require that fish are handled and thereby stressed, oral administration of vaccines as feed supplements is desirable. In addition, in terms of revaccination (boosting) of adult fish held in net pens, oral administration of vaccines is probably the only feasible method to obtain proper protection against diseases over long periods of time. Oral vaccination is considered a suitable method for mass immunization of large and stress-sensitive fish populations. Moreover, oral vaccines may preferably induce mucosal immunity, which is especially important to fish. Experimental oral vaccine formulations include both non-encapsulated and encapsulated antigens, viruses and bacteria. To develop an effective oral vaccine, the desired antigens must be protected against the harsh environments in the stomach and gut so they can remain intact when they reach the lower gut/intestine where they normally are absorbed and transported to immune cells. The most commonly used encapsulation method is the use of alginate microspheres that can effectively deliver vaccines to the intestine without degradation. Other encapsulation methods include chitosan encapsulation, poly D,L-lactide-co-glycolic acid and liposome encapsulation. Only a few commercial oral vaccines are available on the market, including those against infectious pancreatic necrosis virus (IPNV), Spring viremia carp virus (SVCV), infectious salmon anaemia virus (ISAV) and Piscirickettsia salmonis. This review highlights recent developments of oral vaccination in teleost fish.

Intramuscular vaccination of Atlantic lumpfish (Cyclopterus lumpus L.) induces inflammatory reactions and local immunoglobulin M production at the vaccine administration site.

Atlantic lumpfish were vaccinated by intramuscular (im) or intraperitoneal (ip) injection with a multivalent oil-based vaccine, while control fish were injected with phosphate-buffered saline. Four lumpfish per group were sampled for skin/muscle and head kidney tissue at 0, 2, 7, 21 and 42 days post-immunization (dpi) for histopathology and immunohistochemistry (IHC). Gene expressions of secretory IgM, membrane-bound IgM, IgD, TCRα, CD3ε and MHC class IIß were studied in tissues by using qPCR. Im. vaccinated fish showed vaccine-induced inflammation with formation of granulomas and increasing number of eosinophilic granulocyte-like cells over time. On IHC sections, we observed diffuse intercellular staining of secretory IgM at the injection site at 2 dpi, while IgM + cells appeared in small numbers at 21 and 42 dpi. Skin/muscle samples from im. vaccinated fish demonstrated an increase in gene expression of IgM mRNA (secretory and membrane-bound) at 21 and 42 dpi and small changes for other genes. Our results indicated that im. vaccination of lumpfish induced local IgM production at the vaccine injection site, with no apparent proliferation of IgM + cells. Eosinophilic granulocyte-like cells appeared shortly after im. injection and increased in numbers as the inflammation progressed.

Strategies and hurdles using DNA vaccines to fish.

DNA vaccinations against fish viral diseases as IHNV at commercial level in Canada against VHSV at experimental level are both success stories. DNA vaccination strategies against many other viral diseases have, however, not yet yielded sufficient results in terms of protection. There is an obvious need to combat many other viral diseases within aquaculture where inactivated vaccines fail. There are many explanations to why DNA vaccine strategies against other viral diseases fail to induce protective immune responses in fish. These obstacles include: 1) too low immunogenicity of the transgene, 2) too low expression of the transgene that is supposed to induce protection, 3) suboptimal immune responses, and 4) too high degradation rate of the delivered plasmid DNA. There are also uncertainties with regard distribution and degradation of DNA vaccines that may have implications for safety and regulatory requirements that need to be clarified. By combining plasmid DNA with different kind of adjuvants one can increase the immunogenicity of the transgene antigen - and perhaps increase the vaccine efficacy. By using molecular adjuvants with or without in combination with targeting assemblies one may expect different responses compared with naked DNA. This includes targeting of DNA vaccines to antigen presenting cells as a central factor in improving their potencies and efficacies by means of encapsulating the DNA vaccine in certain carriers systems that may increase transgene and MHC expression. This review will focus on DNA vaccine delivery, by the use of biodegradable PLGA particles as vehicles for plasmid DNA mainly in fish.

Adjuvants and immunostimulants in fish vaccines: current knowledge and future perspectives.

Vaccination is the most adequate method to control infectious diseases that threaten the aquaculture industry worldwide. Unfortunately, vaccines are usually not able to confer protection on their own; especially those vaccines based on recombinant antigens or inactivated pathogens. Therefore, the use of adjuvants or immunostimulants is often necessary to increase the vaccine efficacy. Traditional adjuvants such as mineral oils are routinely used in different commercial bacterial vaccines available for fish; however, important side effects may occur with this type of adjuvants. A search for alternative molecules or certain combinations of them as adjuvants is desirable in order to increase animal welfare without reducing protection levels. Especially, combinations that may target specific cell responses and thus a specific pathogen, with no or minor side effects, should be explored. Despite this, the oil adjuvants currently used are quite friendlier with respect to side effects compared with the oil adjuvants previously used. The great lack of fish antiviral vaccines also evidences the importance of identifying optimal combinations of a vaccination strategy with the use of a targeting adjuvant, especially for the promising fish antiviral DNA vaccines. In this review, we summarise previous studies performed with both traditional adjuvants as well as the most promising new generation adjuvants such as ligands for Toll receptors or different cytokines, focussing mostly on their protective efficacies, and also on what is known concerning their effects on the fish immune system when delivered in vivo.

Transgene and immune gene expression following intramuscular injection of Atlantic salmon (Salmo salar L.) with DNA-releasing PLGA nano- and microparticles.

The use of poly-(D,L-lactic-co-glycolic) acid (PLGA) particles as carriers for DNA delivery has received considerable attention in mammalian studies. DNA vaccination of fish has been shown to elicit durable transgene expression, but no reports exist on intramuscular administration of PLGA-encapsulated plasmid DNA (pDNA). We injected Atlantic salmon (Salmo salar L.) intramuscularly with a plasmid vector containing a luciferase (Photinus pyralis) reporter gene as a) naked pDNA, b) encapsulated into PLGA nano- (~320 nm) (NP) or microparticles (~4 µm) (MP), c) in an oil-based formulation, or with empty particles of both sizes. The ability of the different pDNA-treatments to induce transgene expression was analyzed through a 70-day experimental period. Anatomical distribution patterns and depot effects were determined by tracking isotope labeled pDNA. Muscle, head kidney and spleen from all treatment groups were analyzed for proinflammatory cytokines (TNF-α, IL-1ß), antiviral genes (IFN-α, Mx) and cytotoxic T-cell markers (CD8, Eomes) at mRNA transcription levels at days 1, 2, 4 and 7. Histopathological examinations were performed on injection site samples from days 2, 7 and 30. Injection of either naked pDNA or the oil-formulation was superior to particle treatments for inducing transgene expression at early time-points. Empty particles of both sizes were able to induce proinflammatory immune responses as well as degenerative and inflammatory pathology at the injection site. Microparticles demonstrated injection site depots and an inflammatory pathology comparable to the oil-based formulation. In comparison, the distribution of NP-encapsulated pDNA resembled that of naked pDNA, although encapsulation into NPs significantly elevated the expression of antiviral genes in all tissues. Together the results indicate that while naked pDNA is most efficient for inducing transgene expression, the encapsulation of pDNA into NPs up-regulates antiviral responses that could be of benefit to DNA vaccination.

Comparison of Aeromonas salmonicida resistant and susceptible salmon families: a high immune response is beneficial for the survival against Aeromonas salmonicida challenge.

Selective breeding has been employed to improve resistance to infectious diseases in aquaculture and it is of importance to investigate the expression profiles of immune genes together with complement activity of Atlantic salmon with different genetic background in response to pathogens, in particular against Aeromonas salmonicida. This study examined acute phase products, and several central T cell cytokines and a transcription factor in different tissues, namely head kidney, spleen and liver, in two families of Atlantic salmon with high and low mortalities, after challenge by A. salmonicida. The results showed that the expression pattern of target genes differed in lymphoid and non-lymphoid organs in the two families. Generally, in lymphoid organs, higher expression of pro-inflammatory genes, such as TLR5M, TLR5S, GATA3, IFN-γ, IL-17D, as well as the pleiotropic cytokine gene IL-10 in the resistant family was observed at the same time point. One may speculate that a relatively high immune response is a pre-requisite for increased survival in a A. salmonicida challenge test. In addition, the resistant fish possessed higher complement activity pre-challenge compared to susceptible fish. Complement activity may be applied as an indicator in selective breeding for enhanced disease resistance.

Molecular cloning and characterization of Foxp3 in Atlantic salmon (Salmo salar).

Foxp3 is a T cell-specific transcription factor and plays a key role in the development of Treg cells and in the immune regulatory process during inflammation. Here we report cloning and characterization of the full-length cDNA of Atlantic salmon Foxp3, which possesses a Forkhead domain, a zinc finger domain and a leucine-zipper domain as its counterpart in mammals. Foxp3 is highly expressed in thymus. Furthermore, regulated expression was observed in head kidney cells in response to ß-glucan and mitogens (LPS and ConA), and in the head kidney, spleen and liver after intraperitoneal injection of live Aeromonas salmonicida. In addition, transfection of CHSE-214 cells with salmon Foxp3 fused with a C-termial RFP tag, resulted in the expression of the transgene in and close to the nuclei upon stimulation. Taken together, these results suggest the presence of a Foxp3 gene in Atlantic salmon that may be an important transcription factor in immune regulation, and further research may reveal the existence of Treg-like T cells in this species.

Cloning, expression analysis and promoter structure of TBK1 (TANK-binding kinase 1) in Atlantic cod (Gadus morhua L.).

TBK1, also termed NAK or T2K, is a ubiquitous member of the IκB kinase (IKK) family that is required for innate and adaptive immune responses. We have identified and characterized the full-length TBK1 cDNA in Atlantic cod. The cod TBK1 gene consists of 2190 bp open reading frame encoding a polypeptide of 729 amino acids. According to a BLAST search, the cloned TBK1 gene has a high degree of sequence similarity (80.7-92%) to the various members of the TBK1 family, indicating that it is conserved during evolution. RT-PCR showed that the largest quantity of TBK1 transcripts was found in spleen, followed by the liver, gill, head kidney, gut, pyloric caeca, while the expression of TBK1 mRNA in muscle and skin was low. Both PMA, poly I:C and ß-glucan promoted expression of TBK1 transcripts in vivo. Furthermore, we determined an 875 bp sequence upstream of the transcriptional start site (TSS) and found a number of sequence motifs that matched known transcription factor-binding sites. Activities of the presumptive regulatory regions of this gene were assessed by transfecting different 5'-deletion constructs in CHSE-214 cells. After the expression experiments, the results showed that the basal promoters and positive transcriptional regulator activities of cod TBK1 gene were dependent by sequences located from -875 to -425 bp and from -245 to +28 bp upstream of TSS. This study provides further insights into the transcriptional regulation of cod TBK1.

Two-dimensional TIRF-SIM-traction force microscopy (2D TIRF-SIM-TFM).

Quantifying small, rapidly evolving forces generated by cells is a major challenge for the understanding of biomechanics and mechanobiology in health and disease. Traction force microscopy remains one of the most broadly applied force probing technologies but typically restricts itself to slow events over seconds and micron-scale displacements. Here, we improve >2-fold spatially and >10-fold temporally the resolution of planar cellular force probing compared to its related conventional modalities by combining fast two-dimensional total internal reflection fluorescence super-resolution structured illumination microscopy and traction force microscopy. This live-cell 2D TIRF-SIM-TFM methodology offers a combination of spatio-temporal resolution enhancement relevant to forces on the nano- and sub-second scales, opening up new aspects of mechanobiology to analysis.

Overexpression of T-bet, GATA-3 and TGF-ß Induces IFN-γ, IL-4/13A, and IL-17A Expression in Atlantic Salmon.

The overexpression of GATA-3, T-bet and TGF-ß may theoretically induce IL-4/A, IFN-γ and IL-17A expression, respectively. Whether this also applies to fish is not yet known. The plasmid vectors encoding reporter gene (RFP)-tagged T-bet, GATA-3 and TGF-ß were used as overexpression tools, transfected into cells or injected intramuscularly to monitor the expression of IFN-γ, IL-4/13A and IL-17A. In addition, the fish were either experimentally challenged with Vibrio anguillarum (VA group) or Piscirickettsia salmonis (PS group). The reporter gene (RFP) inserted upstream of the GATA-3, T-bet and TGF-ß genes, was observed in muscle cell nuclei and in inflammatory cells after intramuscular (i.m.) injection. PS group: following the injection of GATA-3 and T-bet-encoding plasmids, the expression of GATA-3 and T-bet was high at the injection site. The spleen expression of IFN-γ, following the injection of a T-bet-encoding plasmid, was significantly higher on day 2. VA group: The T-bet and GATA-3-overexpressing fish expressed high T-bet and GATA-3 mRNA levels in the muscles and on day 4 post-challenge. The expression of TGF-ß in the muscles of fish injected with TGF-ß-encoding plasmids was significantly higher on days 7 (8 days pre-challenge) and 19 (4 days after challenge). The protective effects of the overexpression of T-bet, GATA-3 and TGF-ß on both bacterial infections were negligible.

A New IL6 Isoform in Chinese Soft-Shelled Turtle (Pelodiscus sinesis) Discovered: Its Regulation during Cold Stress and Infection.

The Chinese soft-shelled turtle (Pelodiscus sinesis) is a widely cultured commercial species in East and Southeast Asian countries. The turtles frequently suffer from acute cold stress during farming in China. Stress-induced factor such as Interleukin-6 (IL6) is a multifunctional molecule that plays important roles in innate and adaptive immune response. In the present study, we found that the turtle possessed two IL6 transcripts, where one IL6 transcript contained a signal peptide sequence (psIL6), while the other IL6 transcript (psIL6ns) possessed no such signal peptide gene. To test any differential expression of the two isoforms during temperature and microbial stress, turtles were adapted to optimal environmental water temperature (25 °C), stressed by acute cooling for 24 h, followed with the challenge of Aeromonas hydrophila (1.8 × 108 CFU) or Staphylococcus aureus (5.8 × 108 CFU). Gene characterization revealed that psIL6ns, a splicer without codons encoding a signal peptide and identical to the one predicted from genomic sequence, and psIL6, a splicer with codons encoding a signal peptide, were both present. Inducible expression was documented in primary spleen cells stimulated with ConA and poly I: C. The splenic and intestinal expression of psIL6ns and psIL6 was increased in response to temperature stress and bacterial infection.

Interleukin-17D in Atlantic salmon (Salmo salar): molecular characterization, 3D modelling and promoter analysis.

IL-17 is a proinflammatory cytokine that plays an important role in the clearance of extracellular bacteria and contributes to the pathology of many autoimmune and allergic conditions. Much work on IL-17 has been done in humans and higher vertebrates while little work has been conducted in lower vertebrates including fish. In this study, we have cloned and characterized the full-length cDNA and genomic sequence of IL-17D from Atlantic salmon. The Atlantic salmon IL-17D (AsIL-17D) cDNA possessed an open reading frame of 621 bp encoding a putative protein of 206 aa with a predicted molecular weight of 23 kDa. The AsIL-17D gene has two exons and one intron showing the same (genome) organisation compared to zebrafish IL-17D. The encoded protein showed 97.6-48.8% identities to other IL-17D homologues, eight conserved cysteine residues were found within this group. Conserved residues believed to be important in receptor binding were also confirmed in salmon IL-17D by homology modelling. Phylogenetic analysis also confirmed the close relationship with other IL-17D homologues. Functional characterization of the 5' flanking region indicated that the region between -1552 and -150 contained sufficient elements for promoter activity. Tissue expression studies by real-time PCR showed a predominant expression of IL-17D transcript in gonads, skin, intestine, thymus of Atlantic salmon. The involvement of IL-17D during proinflammatory responses was demonstrated by investigating the time-dependent expression profile of IL-17D in head kidney and spleen following intraperitoneal injection of live Aeromonas salmonicida, LPS, and beta-glucan. This study provides further evidence for the existence of distinct homologue of IL-17D isoform in fish showing early expression induced by immunostimulants and bacterial infection that supports the fact that IL-17D is regulated by inflammatory processes in fish.

Bath immunostimulation of rainbow trout (Oncorhynchus mykiss) fry induces enhancement of inflammatory cytokine transcripts, while repeated bath induce no changes.

The application of immunostimulants may be a cost-effective practice in production of delicate and fragile fish fry since it may confer disease resistance. Bath administration to fish fry is considered an ideal delivery route for mass manipulation since there is no need for individual handling. In the current study, rainbow trout fry were bathed with three different immunostimulants: Plasmid DNA, lactoferrin and beta-glucan at two different doses (0.1 microM and 1.0 microM) for 45 min, four times with an interval of 1 week. Ten fish per treatment group were sampled, and RNA of pooled tissues consisting of eye, tongue, skin, gill, thymus, spleen, head kidney, liver, small and large intestine were isolated from the fish obtained at first, second and fourth bathing (24 and 72 h post-bathing). Before cDNA transcription, two parallel samples were pooled giving a total of 5 parallels in each treatment group. Results showed that plasmid DNA and lactoferrin as well as beta-glucan treated fish possessed higher gene expression with regard to the pro-inflammatory cytokines IL-1beta, TNF-alpha, IL-6 and the anti-inflammatory cytokines IL-10 and TGF-beta after the first bathing, especially 24 h post-bathing in the high-dose groups. This indicates that bath delivery of immunostimulants can induce pro-inflammatory responses. No significant changes of the pro-inflammatory cytokine IL-17A transcripts, compared to the respective controls, were observed. Gene expression levels in the immunostimulated fish after the fourth bathing did not show significant differences compared to controls.

Review on Immersion Vaccines for Fish: An Update 2019.

Immersion vaccines are used for a variety of aquacultured fish to protect against infectious diseases caused by bacteria and viruses. During immersion vaccination the antigens are taken up by the skin, gills or gut and processed by the immune system, where the resulting response may lead to protection. The lack of classical secondary responses following repeated immersion vaccination may partly be explained by the limited uptake of antigens by immersion compared to injection. Administration of vaccines depends on the size of the fish. In most cases, immersion vaccination is inferior to injection vaccination with regard to achieved protection. However, injection is problematic in small fish, and fry as small as 0.5 gram may be immersion vaccinated when they are considered adaptively immunocompetent. Inactivated vaccines are, in many cases, weakly immunogenic, resulting in low protection after immersion vaccination. Therefore, during recent years, several studies have focused on different ways to augment the efficacy of these vaccines. Examples are booster vaccination, administration of immunostimulants/adjuvants, pretreatment with low frequency ultrasound, use of live attenuated and DNA vaccines, preincubation in hyperosmotic solutions, percutaneous application of a multiple puncture instrument and application of more suitable inactivation chemicals. Electrostatic coating with positively charged chitosan to obtain mucoadhesive vaccines and a more efficient delivery of inactivated vaccines has also been successful.

Trained Innate Immunity of Fish Is a Viable Approach in Larval Aquaculture.

The general understanding has been that only adaptive immunity is capable of immunological memory, but this concept has been challenged in recent years by studies showing that innate immune systems can mount resistance to reinfection-as the innate immune system can adapt its function following an insult. Innate immune training offers an attractive approach in intensive fish larval rearing, especially since the adaptive immune system is not fully developed. Trained innate immunity will potentially favor robust fish in terms of resistance to viral and bacterial diseases. So-called immunostimulants such as ß-glucans have for decades been used both in laboratories and in intensive fish aquaculture. Treatment of fish by ß-glucans (and by other substances with pathogen-associated molecular patterns) often induces activation of non-specific/innate immune mechanisms and induces higher disease resistance. The reported effects of e.g., ß-glucans fit nicely into the concept "trained innate immunity," but the research on fish does not yet include analysis of epigenetic changes that may be a prerequisite for long-lasting trained innate immunity. In this "perspective," we will discuss how in practical terms and based on prior knowledge one can introduce innate immune training in brood stock fish, and their offspring, and whether innate immune training by ß-glucans is a viable approach in larval aquaculture.