The Effect of an Attenuated Live Vaccine against Salmonid Rickettsial Septicemia in Atlantic Salmon (Salmo salar) Is Highly Dependent on Water Temperature during Immunization.

Salmonid Rickettsial Septicemia (SRS), caused by the bacterium Piscirickettsia salmonis, is the main reason for antibiotic usage in the Chilean aquaculture industry. In 2016, a live attenuated vaccine (ALPHA JECT LiVac® SRS, PHARMAQ AS) was licensed in Chile and has been widely used in farmed salmonids since then. In experimental injection and cohabitation laboratory challenge models, we found that the vaccine is effective in protecting Atlantic salmon (Salmo salar) for at least 15 months against P. salmonis-induced mortality. However, the protection offered by the vaccine is sensitive to temperature during immunization. Fish vaccinated and immunized at 10 °C and above were well protected, but those immunized at 7 °C and 8 °C (the lower end of the temperature range commonly found in Chile) experienced a significant loss of protection. This temperature-dependent loss of effect correlated with the amount of vaccine-strain RNA detected in the liver the first week after vaccination and with in vitro growth curves, which failed to detect any growth at 8 °C. We found that good vaccine efficacy can be restored by exposing fish to 15 °C for the first five days after vaccination before lowering the temperature to 7 °C for the remaining immunization period. This suggests that maintaining the correct temperature during the first few days after vaccination is crucial for achieving a protective immune response with ALPHA JECT LiVac® SRS. Our results emphasize the importance of temperature control when vaccinating poikilothermic animals with live vaccines.

Novel mannose binding natterin-like protein in the skin mucus of Atlantic cod (Gadus morhua).

This study presents the first report of purification of natterin-like protein (Nlp) in a non-venomous fish. The peptide identities of purified cod Nlp were confirmed through LC-MSMS and matched to a cod expressed sequence tag (EST). A partial cod nlp nucleotide sequence was amplified and sequenced based on this EST. Multiple sequence alignment of cod Nlp showed considerable homology with other teleost Nlps and the presence of an N-terminal jacalin-like lectin domain coupled with a C-terminal toxin domain. nlp expression was higher in skin, head kidney, liver and spleen than in other tissues studied. Hemaggluttination of horse red blood cells by Nlp was calcium dependent and inhibited by mannose. A Vibrio anguillarum bath challenge however, did not alter the expression of cod nlp transcripts in the skin and gills. Further functional characterization is required to establish the significance of this unique protein in Atlantic cod and other teleosts.

Passive Immunization of Farmed Fish.

The fast growth and potential of global aquaculture has necessitated the adoption of sustainable and welfare-oriented therapeutics and prophylactic strategies. Knowledge gathered from studies about maternal passive immunity in fish and fish-to-fish passive immunization experiments supports the concept of using therapeutic Abs (of piscine and other vertebrate origin) in aquaculture. Traditional Ab formats (IgG, IgM) are expensive and laborious to produce; however, the introduction of new rAb fragments and single-domain Abs have reinvigorated the concept of passive immunization. This review will focus primarily on farmed salmonids (salmon and trout) within a comparative context and will give an overview of the basic principles and scientific premises for the passive immunization strategy, including existing and emerging Ab therapeutics.

Dietary Yeast Cell Wall Extract Alters the Proteome of the Skin Mucous Barrier in Atlantic Salmon (Salmo salar): Increased Abundance and Expression of a Calreticulin-Like Protein.

In order to improve fish health and reduce use of chemotherapeutants in aquaculture production, the immunomodulatory effect of various nutritional ingredients has been explored. In salmon, there is evidence that functional feeds can reduce the abundance of sea lice. This study aimed to determine if there were consistent changes in the skin mucus proteome that could serve as a biomarker for dietary yeast cell wall extract. The effect of dietary yeast cell wall extract on the skin mucus proteome of Atlantic salmon was examined using two-dimensional gel electrophoresis. Forty-nine spots showed a statistically significant change in their normalised volumes between the control and yeast cell wall diets. Thirteen spots were successfully identified by peptide fragment fingerprinting and LC-MS/MS and these belonged to a variety of functions and pathways. To assess the validity of the results from the proteome approach, the gene expression of a selection of these proteins was studied in skin mRNA from two different independent feeding trials using yeast cell wall extracts. A calreticulin-like protein increased in abundance at both the protein and transcript level in response to dietary yeast cell wall extract. The calreticulin-like protein was identified as a possible biomarker for yeast-derived functional feeds since it showed the most consistent change in expression in both the mucus proteome and skin transcriptome. The discovery of such a biomarker is expected to quicken the pace of research in the application of yeast cell wall extracts.

Differentially expressed proteins in the skin mucus of Atlantic cod (Gadus morhua) upon natural infection with Vibrio anguillarum.

BACKGROUND: Vibriosis caused by V. anguillarum is a commonly encountered disease in Atlantic cod farms and several studies indicate that the initiation of infection occurs after the attachment of the pathogen to the mucosal surfaces (gut, skin and gills) of fish. Therefore it is necessary to investigate the role of different mucosal components in fish upon V. anguillarum infection. The present study has two parts; in the first part we analyzed the differential expression of skin mucus proteins from Atlantic cod naturally infected with V. anguillarum using two dimensional gel electrophoresis coupled with mass spectrometry. In the second part, a separate bath challenge experiment with V. anguillarum was conducted to assess the mRNA levels of the genes in skin tissue, corresponding to the selected proteins identified in the first part. RESULTS: Comparative proteome analysis of skin mucus of cod upon natural infection with V. anguillarum revealed key immune relevant proteins like calpain small subunit 1, glutathione-S-transferase omega 1, proteasome 26S subunit, 14-kDa apolipoprotein, beta 2-tubulin, cold inducible RNA binding protein, malate dehydrogenase 2 (mitochondrial) and type II keratin that exhibited significant differential expression. Additionally a number of protein spots which showed large variability amongst individual fish were also identified. Some of the proteins identified were mapped to the immunologically relevant JNK (c-Jun N-terminal kinases) signalling pathway that is connected to cellular events associated with pathogenesis. A bath challenge experiment with V. anguillarum showed differential expression of beta 2-tubulin, calpain small subunit 1, cold inducible RNA binding protein, flotillin1, and glutathione S-transferase omega 1 transcripts in the skin tissue of cod during early stages of infection. CONCLUSIONS: Differentially expressed proteins identified in the cod skin mucus point towards their possible involvement in V. anguillarum pathogenesis. The role of some of these proteins in vibriosis in cod described in this paper can be considered unconventional with respect to their established functions in higher vertebrates. Based on the differential expression of these proteins they are possibly important components of fish defence against bacteria and innate immunity at large. The feasibility of utilizing these proteins/genes as markers of bacterial infection or stress in cod needs to be explored further.

Localization and functional properties of two galectin-1 proteins in Atlantic cod (Gadus morhua) mucosal tissues.

Galectin-1 is a ß-galactoside binding lectin with multiple immune functions in higher vertebrates. We report the characterization of two galectin-1 proteins from Atlantic cod, with emphasis on mucosal tissues. Tissue distribution of these two ≈14kDa galectin-1 proteins (Codgal1-1 and Codgal1-2) was ascertained by western blotting of one dimensional (1D) and two dimensional (2DE) gels. The two galectin-1 proteins were differentially localized in the mucosal tissues of cod. Codgal1-1 was predominantly localized in the basal cells of skin and this protein was present in all the early developmental stages examined, indicating a likely involvement in developmental processes. The two lectins were also localized in the adherent macrophage-like cells (MLC) from cod head kidney and results gathered indicate their possible secretion during Francisella noatunensis infection, suggesting that they are active components of immune defence. Lactose affinity chromatography coupled with gel filtration co-purified the two cod galectin-1 proteins, which hemagglutinated horse red blood cells in a lactose inhibitable manner. They also could bind and agglutinate both Gram-positive and Gram-negative bacteria. This study suggests multiple functional roles for galectin-1, especially in development and innate immune response of Atlantic cod.

Proteome reference map of the skin mucus of Atlantic cod (Gadus morhua) revealing immune competent molecules.

The skin mucosal proteome of Atlantic cod (Gadus morhua) was mapped using a 2D PAGE, LC-MS/MS coupled approach. Mucosal proteins from naive fish were identified primarily by similarity searches across various cod EST databases. The identified proteins were clustered into 8 groups based on gene ontology classification for biological process. Most of the proteins identified from the gel are hitherto unreported for cod. Galectin-1, mannan binding lectin (MBL), serpins, cystatin B, cyclophilin A, FK-506 binding protein, proteasome subunits (alpha-3 and -7), ubiquitin, and g-type lysozyme are considered immune competent molecules. Five of the aforementioned proteins were cloned and their tissue distribution was analysed by RT-PCR.

Characterization of GP21 and GP12: Two Potential Probiotic Bacteria Isolated from the Gastrointestinal Tract of Atlantic Cod.

This study evaluated the probiotic potential of GP21 (Pseudomonas sp.) and GP12 (Psychrobacter sp.), two bacteria isolated from the intestinal tract of a cold-water fish, Atlantic cod. The antagonistic activity of the two intestinal bacteria against two fish pathogens (Vibrio anguillarum and Aeromonas salmonicida subsp. salmonicida) was studied under different physical conditions. Further, their resistance to physiological barriers and their ability to form biofilms were examined. In addition, a test was conducted to confirm that the isolates were not pathogenic to the host fish. The two bacteria exhibited differences in their antagonism to the pathogens. Both were active against V. anguillarum at mildly acidic conditions over a 5-day period. The activity of GP21 against A. salmonicida was greater at pH 7-8. The maximum antagonistic activity was observed at a temperature of 15°C and at a salt concentration of 15 ppt for both the isolates. They did not produce acids, could release siderophores and tolerated both the acidic environment and the bile salts. Their ability to form biofilms was high around 15°C and when iron was supplemented in the medium at 5 µmol l(-1). There was no mortality of fish during the pathogenicity experiment, confirming the safety of both isolates for further applications. Considering the favorable characteristics identified here, it could be concluded that GP21 and GP12 isolated from the gastrointestinal tract of Atlantic cod are potential probiotic candidates.