Growth hormone (GH) and growth hormone release factor (GRF) modulate the immune response in the SHK-1 cell line and leukocyte cultures of head kidney in Atlantic salmon.

To clarify the role of growth hormone (GH) in the immune system of fish, we examine the comparative effect of GH and Growth Hormone Release Factor (GRF) on leukocytes culture of the head kidney of Atlantic salmon and the SHK-1 cell line. There are studies that associate the growth hormone (GH) / insulin-like growth factor (IGF) axis with the immune regulation of fish. However, there is no evidence that GH and GRF stimulate Atlantic salmon leukocyte cell lines, where there arent reports on expression changes in different immune response markers. Thus, we investigated the effect of GH and GRF in Atlantic salmon leukocytes extracted from head kidney and the SHK-1 cell line on the different immune response markers such as: NLRC5, NLRC3, IL-1ß, TNF-α, and IL-8 through qPCR. Our data suggest that GH increases the expression of NLRC5, NLRC3, and IL-1ß mainly at 16 h post-stimulation in Atlantic salmon leukocytes. This indicates differential regulation between the two models used, helping us to better understand the independent action of GH on the immune system and the GH / IGF axis for future research.

Cellular stress responses of Eleginops maclovinus fish injected with Piscirickettsia salmonis and submitted to thermal stress.

Fluctuations in ambient temperature along with the presence of pathogenic microorganisms can induce important cellular changes that alter the homeostasis of ectothermic fish. The aim of this study was to evaluate how sudden or gradual changes in environmental temperature together with the administration of Piscirickettsia salmonis modulate the transcription of genes involved in cellular stress response in the liver of Eleginops maclovinus. Fish were subjected to the following experimental conditions in duplicate: C- 12 °C: Injection only with culture medium, C+ 12 °C: Injection with P. salmonis, AM 18 °C: Injection only with culture medium under acclimation at 18 °C, AB 18 °C: Injection with P. salmonis under acclimation at 18 °C, SM 18 °C: Injection only with culture medium and thermal shock at 18 °C and SB 18 °C: Injection with P. salmonis and thermal shock at 18 °C and sampling at 4-, 8-, 12-, 16- and 20-day post injection (dpi). The genes implied in the heat shock response (HSP70, HSC70, HSP90, and GRP78), apoptosis pathway (BAX and SMAC/Diablo), ubiquitination (E2, E3, ubiquitin, and CHIP), and 26 proteasome complex (PSMB7, PSMC1, and PSMA2) showed expression profiles dependent on time and type of injection applied. All the genes greatly increased their expression levels at day 16 and showed moderate increases at day 20, except for PSMA2 which showed a higher increase between 4- and 12-day post challenges. Our results suggest that the changes observed at the final days of the experiment are due to temperature more than P. salmonis.

High doses of Francisella noatunensis induces an immune response in Eleginops maclovinus.

Francisella noatunensis subsp. noatunensis, the etiological agent of Francisellosis, affects a large number of farmed species such as Salmo salar. This species coexists with several native species in the same ecosystem, including Eleginops maclovinus. Our objective was to evaluate the susceptibility, presence of clinical symptoms, and the ability of Eleginops maclovinus to respond to Francisella infection. For this, healthy individuals were inoculated with 1.5 × 101, 1.5 × 105, and 1.5 × 1010 bact/µL of Francisella by intraperitoneal injection, subsequently the fish were sampled on days 1, 3, 7, 14, 21, and 28 post injection (dpi). At the end of the experiment, no mortality, nor internal and external clinical signs were observed, although in the high dose anaemia was detected. Additionally, bacteria were detected in all three doses, however there was replication at day 28 only in the liver in the high dose. Analysis of gene expression by qPCR showed that the spleen generated an immune response against infection from day 1 dpi, however at day 7 dpi most of the genes suffered repressed expression; observing over expression of the genes C3, NLRC3, NLRC5, MHCI, IgM. In contrast, expression in the anterior kidney did not vary significantly during the challenge. IgM quantification showed the production of antibodies in the medium and high doses. This study provides new knowledge about Francisella infection and the long-lasting and specific immune response generated by Eleginops maclovinus. It also demonstrates its susceptibility to Francisellosis where there is a difference in the immune response according to the tissue.

The expression pattern of calcium signaling-related genes during smoltification of Salmo salar in productive conditions.

Variations in the mRNA expression of hepatic and muscle genes that are related to calcium signaling were analyzed by real-time qPCR in farmed Atlantic salmon (Salmo salar L. 1758) to determine changes in expression between parr and smolt stages. These organs were selected due to their close relationship with calcium signaling and metabolism (e.g., glycolysis, oxidative phosphorylation, muscle contraction). Differential expression between smolt and parr specimens and between organs was observed. Compared to parr specimens, smolts exhibited upregulated expression of the calcitonin receptor precursor, calcitonin receptor, calcitonin isoform, parathyroid hormone, and calmodulin in the liver. This pattern was inverse in muscle, with the exception of calmodulin, which was significantly upregulated in smolts compared to parr. Additionally, plasma calcium was decreased in the smolt condition. This study is the first to characterize the expression pattern of calcium signaling-related genes in the liver and muscle of parr and smolt S. salar. However, further functional studies are required to obtain a wider understanding about the physiological changes that accompany the productive conditions during smoltification.

Transcriptional activation of genes involved in oxidative stress in Salmo salar challenged with Piscirickettsia salmonis.

Piscirickettsiosis caused by Piscirickettsia salmonis constitutes one of the main problems in farmed salmonid and marine fishes. The objective of this study was to evaluate the modulation of genes involved in the oxidative stress in the liver and muscle of Salmo salar challenge with low dosage of P. salmonis. The treatment (in duplicate) were as follows: Control injection (culture medium) and P. salmonis injection (1 × 102 PFU/mL) with sampling (liver and muscle) at several time-points during the 42-days experimental period (dpi). In liver, the gene expression of superoxide dismutase (SOD) and acetylcholinesterase (AChE) had differences with the control group only at 7 dpi, compared with glutathione-S-transferase (GST) and heat shock protein 70 (HSP70) that presented increases at 7 and 21 dpi. The glutathione peroxidase (GPx) and catalase (CAT) mRNAs were elevated at 13 and 21 dpi, respectively. While glutathione reductase (GR) and cytochrome P450 (P450) did not show variations in their expression during the experimental course. In muscle, the expression of CAT and AChE was higher than in the control condition at 2 and 42 dpi, respectively. While the number of transcripts SOD, GPx, GR, GST, P450 and HSP70 showed increases at 7- and 42-days post injection. The results suggest a transcriptional activation of genes involved in oxidative stress in both liver and muscle, with expression profiles that were tissue-specific and dependent on the time. This is the first study that reveals the transcriptional participation of all these genes associated with oxidative stress in response to the injection of P. salmonis.

Temperature modulates the immunological response of the sub-antarctic notothenioid fish Eleginops maclovinus injected with Piscirickettsia salmonis.

Eleginops maclovinus is a eurythermic fish that under natural conditions lives in environments with temperatures ranging from 4 to 18 °C and can be usually captured near salmon farming areas. The aim of this study was to evaluate the effect of temperature over the innate and adaptive immune response of E. maclovinus challenged with Piscirickettsia salmonis following different treatments: C (control injection with culture medium at 12 °C), C+ (bacterial injection at 12 °C), 18 °C c/A + B (injection with culture medium in acclimation at 18 °C), 18 °C c/A + B (bacterial injection in acclimation at 18 °C), 18 °C s/A + M (injection with culture medium without acclimation at 18 °C) and 18 °C s/A + B (bacterial injection without acclimation at 18 °C). Each injection had 100 µL of culture medium or with 100 µL at a concentration 1 × 108 of live bacteria, sampling six fish per group at 4, 8, 12, 16 and 20 days post-injection (dpi). Expression of the mRNA related with the innate immune response gene (TLR1, TLR5, TLR8, NLRC3, NLRC5, MyD88 and IL-1ß) as well as the adaptive immune response gene (MHCI, MHCII, IgMs and IgD) were measured in spleen and head kidney. Gene expression profiles were treatment-type and time dependent. Levels of Immunoglobulin M (IgM) increased in challenged groups with P. salmonis from day 8-20 post challenge, which suggest activation of B cells IgM + through P. salmonis epitope detection. Additionally, a rise in temperature from 12 °C (C+) to 18 °C (with/without acclimation) also resulted in antibody increment detected in serum with significant differences between "18 °C c/A + B" and "18 °C s/A + B" groups. This is the first study that evaluates the effect of temperature changes and mRNA expression related with immune system gene over time on E. maclovinus, a native wild life fish that cohabits in the salmon farming environment.

Immunological response of the Sub-Antarctic Notothenioid fish Eleginops maclovinus injected with two strains of Piscirickettsia salmonis.

Eleginops maclovinus is an endemic fish to Chile that lives in proximity to salmonid culture centers, feeding off of uneaten pellet and salmonid feces. Occurring in the natural environment, this interaction between native and farmed fish could result in the horizontal transmission of pathogens affecting the aquaculture industry. The aim of this study was to evaluate the innate and adaptive immune responses of E. maclovinus challenged with P. salmonis. Treatment injections (in duplicate) were as follows: control (100 µL of culture medium), wild type LF-89 strain (100 µL, 1 × 108 live bacteria), and antibiotic resistant strain Austral-005 (100 µL, 1 × 108 live bacteria). The fish were sampled at various time-points during the 35-day experimental period. The gene expression of TLRs (1, 5, and 8), NLRCs (3 and 5), C3, IL-1ß, MHCII, and IgMs were significantly modulated during the experimental period in both the spleen and gut (excepting TLR1 and TLR8 spleen expressions), with tissue-specific expression profiles and punctual differences between the injected strains. Anti-P. salmonis antibodies increased in E. maclovinus serum from day 14-28 for the LF-89 strain and from day 14-35 for the Austral-005 strain. These results suggest temporal activation of the innate and adaptive immune responses in E. maclovinus tissues when injected by distinct P. salmonis strains. The Austral-005 strain did not always cause the greatest increases/decreases in the number of transcripts, so the magnitude of the observed immune response (mRNA) may not be related to antibiotic resistance. This is the first immunological study to relate a pathogen widely studied in salmonids with a native fish.

Ectoparasite Caligus rogercresseyi modifies the lactate response in Atlantic salmon (Salmo salar) and Coho salmon (Oncorhynchus kisutch).

Although Caligus rogercresseyi negatively impacts Chilean salmon farming, the metabolic effects of infection by this sea louse have never been completely characterized. Therefore, this study analyzed lactate responses in the plasma, as well as the liver/muscle lactate dehydrogenase (LDH) activity and gene expression, in Salmo salar and Oncorhynchus kisutch infested by C. rogercresseyi. The lactate responses of Atlantic and Coho salmon were modified by the ectoparasite. Both salmon species showed increasing in plasma levels, whereas enzymatic activity increased in the muscle but decreased in the liver. Gene expression was overexpressed in both Coho salmon tissues but only in the liver for Atlantic salmon. These results suggest that salmonids need more energy to adapt to infection, resulting in increased gene expression, plasma levels, and enzyme activity in the muscles. The responses differed between both salmon species and over the course of infection, suggesting potential species-specific responses to sea-lice infection.

Identification, characterization and modulation of ferritin-H in the sub-Antarctic Notothenioid Eleginops maclovinus challenged with Piscirickettsia salmonis.

Ferritin is a major iron storage protein essential not only in the infectious process, but also in any circumstance generating oxidative stress. In this study, the cDNA coding sequence of ferritin-H was obtained from the sub-Antarctic Notothenioid fish Eleginops maclovinus through transcriptomic analysis of the head kidney. This sequence contained a 534 bp open reading frame that coded for a 177 amino acid protein with a molecular weight of 20,786.2 Da and a theoretical pI of 5.56. The protein displayed a region of iron putative response elements in the 5'UTR, two putative ferritin iron-binding region signatures, and seven characteristic amino acids with ferroxidase functions. Phylogenetic analysis related this sequence to ferritin-H sequences of other Antarctic Notothenioid fish, sharing 96.61% similarity. Constitutive gene expression analysis in different organs revealed increased ferritin-H gene expression in the gills, spleen, muscle, and liver. After infection with two bacterial strains of Piscirickettsia salmonis (LF-89 and Austral-005), ferritin-H was differentially expressed depending on bacterial strain and tissue. This study provides relevant information towards understanding the iron metabolism of a sub-Antarctic Notothenioid fish.

Effects on the metabolism, growth, digestive capacity and osmoregulation of juvenile of Sub-Antarctic Notothenioid fish Eleginops maclovinus acclimated at different salinities.

In this study we assessed the influence of three different environmental salinities (5, 15 and 31 psu during 90 days) on growth, osmoregulation, energy metabolism and digestive capacity in juveniles of the Notothenioid fish Eleginops maclovinus. At the end of experimental time samples of plasma, liver, gill, intestine, kidney, skeletal muscle, stomach and pyloric caeca were obtained. Growth, weight gain, hepatosomatic index and specific growth rate increased at 15 and 31 psu and were lower at 5 psu salinity. Gill Na(+), K(+)-ATPase (NKA) activity presented a "U-shaped" relationship respect to salinity, with its minimum rates at 15 psu, while this activity correlated negatively with salinity at both anterior and posterior intestinal portions. No significant changes in NKA activity were observed in kidney or mid intestine. Large changes in plasma, metabolite levels and enzymatic activities related to energy metabolism in liver, gill, intestine, kidney and muscle were generally found in the groups exposed to 5 and 31 psu compared to the 15 psu group. Only the pepsin activity (digestive enzymes) assessed enhanced with environmental salinity, while pyloric caeca trypsin/chymotrypsin ratio decreased. This study suggests that juvenile of E. maclovinus presents greater growth near its iso-osmotic point (15 psu) and hyperosmotic environment (31 psu). Acclimation to low salinity increased the osmoregulatory expenditure as seen by the gill and anterior intestine results, while at high salinity, branchial osmoregulatory activity was also enhanced. This requires the mobilization of lipid stores and amino acids, thereby holding the growth of fish back. The subsequent reallocation of energy sources was not sufficient to maintain the growth rate of fish exposed to 5 psu. Thus, E. maclovinus juveniles present better growth efficiencies in salinities above the iso-osmotic point and hyperosmotic environment of this species, showing their best performance at 15 psu as seen by the main osmoregulatory and energy metabolism enzymatic activities.

Effectiveness of egg yolk immunoglobulin against the intracellular salmonid pathogen Piscirickettsia salmonis.

AIMS: To produce and characterize egg yolk immunoglobulin (IgY) against the fish intracellular pathogen Piscirickettsia salmonis as well as to evaluate the antibacterial activity of IgY in vitro and the availability in the serum of fish immunized orally. METHODS AND RESULTS: Specific IgY was produced by immunizing hens with P. salmonis proteins. The IgY was obtained from egg yolks using the ammonium sulphate precipitation method and it was characterized by SDS-PAGE, Western-blot and ELISA, demonstrating that anti-P. salmonis IgY strongly reacted specifically against P. salmonis proteins. In an in vitro neutralization assay, IgY inhibited the growth of P. salmonis in liquid medium at concentrations ranging from 128 to 256 µg ml(-1) in a dose-dependent manner. Interestingly, IgY against P. salmonis also generates a strong protective effect on the infection of P. salmonis in salmon head kidney-1 cells. In addition, the bacteriostatic function of IgY appears to result possibly from agglutination by the interaction of IgY with surface components of the pathogen. Finally, to confirm this IgY as an alternative for salmonid treatment, Atlantic salmon (Salmo salar) specimens were orally inoculated with IgY. The analysis of the sera demonstrates that IgY was effectively transported by fish intestine and that this immunoglobulins maintains its properties and recognizes several proteins of P. salmonis up to 12 h after inoculation of IgY against P. salmonis. CONCLUSIONS: Specific IgY effectively inhibited the growth of P. salmonis and this immunoglobulin can be released in the Atlantic salmon sera when administered orally to fish. SIGNIFICANCE AND IMPACT OF THE STUDY: We propose that this specific IgY against this fastidious micro-organism could be a useful strategy for the treatment of piscirickettsiosis.