Current vaccination strategy against Piscirickettsia salmonis in Chile based only on the EM-90 genogroup shows incomplete cross-protection for the LF-89 genogroup.

Piscirickettsia salmonis, the primary bacterial disease in Chilean salmon farming, necessitates a constant refinement of control strategies. This study hypothesized that the current vaccination strategy for SRS control in the Chilean Atlantic salmon aquaculture industry, which has been in place since 2017 (ALPHA JECT® 5.1 plus LiVac®), solely relies on vaccines formulated with the EM-90 genogroup of P. salmonis (PS-EM-90), triggering a partial cross-immunity response in fish infected with the LF-89 genogroup (PS-LF-89). Relative Percent Survival (RPS) and cell-mediated immune (CMI) response were evaluated in Atlantic salmon post-smolts vaccinated with the standard vaccination strategy but challenged with both PS-EM-90 and PS-LF-89, in addition to other vaccination strategies considering primo vaccination and booster with other commercial vaccines and the possible enhancing effects of the combination with a natural immunomodulator (PAQ-Xtract®) administered orally. The intraperitoneal (I.P.) challenge was performed after 2395°-days (DD) after the start of the immunostimulant delivery, 1905 DD after the primo vaccination, and 1455 DD after the booster vaccination. Unvaccinated fish showed 73.6 and 41.7 % mortality when challenged with PS-EM-90 and PS-LF-89, respectively. Fish infected with PS-LF-89 died significantly faster (21 days post-infection, dpi) than fish challenged with PS-EM-90 (28 dpi) (p = 0.0043) and had a higher probability of death (0.4626) than fish challenged with PS-EM-90. RPS had a significant positive correlation with the PS-EM-90 load of the P. salmonis genogroup (r = 0.540, p < 0.01) but not with the PS-LF-89 load (r = 0.155, p > 0.05). This demonstrated that the immunization strategies were more effective in lowering PS-EM-90 loads, resulting in higher survival rates in fish challenged with PS-EM-90. The current industry vaccination strategy recorded a 100 % RPS when fish were challenged with PS-EM-90, but the RPS dropped significantly to 77 % when fish were challenged with PS-LF-89, meaning that the strategy did not show complete cross-protection. But after adding PAQ-Xtract®, the RPS improved from 77 % to 92 % in fish that were vaccinated with the standard method but then challenged with PS-LF-89. The most effective vaccination strategy was based on LiVac® as primo vaccination and ALPHA JECT® 5.1 plus LiVac® as booster vaccination, with or without PAQ-Xtract®, in both PS-EM-90 (100 %) and PS-LF-89 (96 %) challenged fish. The serum concentration of anti-P. salmonis IgM did not show a correlation with the protection of immunization strategies expressed in survival. Low serum IL-12 and high serum IFNγ concentrations showed a correlation with higher bacterial loads and lower survival. Aggregate analysis showed a significant correlation between higher numbers of CD8+ cells in the head-kidney, higher fish survival, and a lower bacterial load. The immunization strategies were safe for fish and induced only mild microscopic lesions in the gut. Taken together, our results help to better understand the biological interaction between P. salmonis and post-smolt vaccinated Atlantic salmon to deepen the knowledge on vaccine-induced protection, CMI immune response, and cross-immunity applied to improve the current immunization strategy for SRS control in the Chilean salmon industry.

Flavobacterium facile sp. nov., isolated from water system of Atlantic salmon (Salmo salar) fry cultured in Chile.

Strain T-12T, an orange, Gram-stain-negative, non-motile, rod-shaped strain, was isolated in November 2013 from water samples collected from an Atlantic salmon (Salmo salar) fry culturing system at a fish farm in Chile. Phylogenetic analysis based on 16S rRNA sequences (1394 bp) revealed that strain T-12T belonged to the genus Flavobacterium, showing close relationships to Flavobacterium bernardetii F-372T (99.48 %) and Flavobacterium terrigena DS-20T (98.50 %). The genome size of strain T-12T was 3.28 Mb, with a G+C content of 31.1 mol%. Genome comparisons aligned strain T-12T with Flavobacterium bernardetii F-372T (GCA_011305415) and Flavobacterium terrigena DSM 17934T (GCA_900108955). The highest digital DNA-DNA hybridization (dDDH) values were 42.6 % with F. bernardetii F-372T (GCA_011305415) and 33.9 % with F. terrigena DSM 17934T (GCA_900108955). Pairwise average nucleotide identity (ANI) calculations were below the species cutoff, with the best results with F. bernardetii F-372T being: ANIb, 90.33 %; ANIm, 91.85 %; and TETRA, 0.997 %. These dDDH and ANI results confirm that strain T-12T represents a new species. The major fatty acids were iso-C15 : 0 and C15 : 1ω6с. Detected polar lipids included phospholipids (n=2), aminophospholipid (n=1), aminolipid (n=1) and unidentified lipids (n=2). The predominant respiratory quinone was menaquinone MK7 (80 %) followed by MK-6 (20 %). Phenotypic, chemotaxonomic, and genomic data support the classification of strain T-12T (=CECT 30410T=RGM 3222T) as representing a novel species of Flavobacterium, for which the name Flavobacterium facile sp. nov. is proposed.

Discovery and Characterization of the ddx41 Gene in Atlantic Salmon: Evolutionary Implications, Structural Functions, and Innate Immune Responses to Piscirickettsia salmonis and Renibacterium salmoninarum Infections.

The innate immune response in Salmo salar, mediated by pattern recognition receptors (PRRs), is crucial for defending against pathogens. This study examined DDX41 protein functions as a cytosolic/nuclear sensor for cyclic dinucleotides, RNA, and DNA from invasive intracellular bacteria. The investigation determined the existence, conservation, and functional expression of the ddx41 gene in S. salar. In silico predictions and experimental validations identified a single ddx41 gene on chromosome 5 in S. salar, showing 83.92% homology with its human counterpart. Transcriptomic analysis in salmon head kidney confirmed gene transcriptional integrity. Proteomic identification through mass spectrometry characterized three unique peptides with 99.99% statistical confidence. Phylogenetic analysis demonstrated significant evolutionary conservation across species. Functional gene expression analysis in SHK-1 cells infected by Piscirickettsia salmonis and Renibacterium salmoninarum indicated significant upregulation of DDX41, correlated with increased proinflammatory cytokine levels and activation of irf3 and interferon signaling pathways. In vivo studies corroborated DDX41 activation in immune responses, particularly when S. salar was challenged with P. salmonis, underscoring its potential in enhancing disease resistance. This is the first study to identify the DDX41 pathway as a key component in S. salar innate immune response to invading pathogens, establishing a basis for future research in salmonid disease resistance.

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.

Flavobacterium psychraquaticum sp. nov., isolated from water system of Atlantic salmon (Salmo salar) smolts cultured in Chile.

Strain LB-N7T, a novel Gram-negative, orange, translucent, gliding, rod-shaped bacterium, was isolated from water samples collected from an open system of Atlantic salmon (Salmo salar) smolts in a fish farm in Chile during a flavobacterial infection outbreak in 2015. Phylogenetic analysis based on 16S rRNA sequences (1337 bp) revealed that strain LB-N7T belongs to the genus Flavobacterium and is closely related to the type strains Flavobacterium ardleyense A2-1T (98.8 %) and Flavobacterium cucumis R2A45-3T (96.75 %). The genome size of strain LB-N7T was 2.93 Mb with a DNA G+C content 32.6 mol%. Genome comparisons grouped strain LB-N7T with Flavobacterium cheniae NJ-26T, Flavobacterium odoriferum HXWNR29T, Flavobacterium lacisediminis TH16-21T and Flavobacterium celericrescens TWA-26T. The calculated digital DNA-DNA hybridization values between strain LB-N7T and the closest related Flavobacterium strains were 23.3 % and the average nucleotide identity values ranged from 71.52 to 79.39 %. Menaquinone MK-6 was the predominant respiratory quinone, followed by MK-7. The major fatty acids were iso-C15 : 0 and anteiso-C15 : 0. The primary polar lipids detected included nine unidentified lipids, two amounts of aminopospholipid and phospholipids, and a smaller amount of aminolipid. Phenotypic, genomic, and chemotaxonomic data suggest that strain LB-N7T (=CECT 30406T=RGM 3221T) represents as a novel bacterial species, for which the name Flavobacterium psychraquaticum sp. nov. is proposed.

[Cu(NN1)2]ClO4, a Copper (I) Complex as an Antimicrobial Agent for the Treatment of Piscirickettsiosis in Atlantic Salmon.

Piscirickettsia salmonis is the pathogen that most affects the salmon industry in Chile. Large quantities of antibiotics have been used to control it. In search of alternatives, we have developed [Cu(NN1)2]ClO4 where NN1 = 6-((quinolin-2-ylmethylene)amino)-2H-chromen-2-one. The antibacterial capacity of [Cu(NN1)2]ClO4 was determined. Subsequently, the effect of the administration of [Cu(NN1)2]ClO4 on the growth of S. salar, modulation of the immune system and the intestinal microbiota was studied. Finally, the ability to protect against a challenge with P. salmonis was evaluated. The results obtained showed that the compound has an MIC between 15 and 33.9 µg/mL in four isolates. On the other hand, the compound did not affect the growth of the fish; however, an increase in the transcript levels of IFN-γ, IL-12, IL-1ß, CD4, lysozyme and perforin was observed in fish treated with 40 µg/g of fish. Furthermore, modulation of the intestinal microbiota was observed, increasing the genera of beneficial bacteria such as Lactobacillus and Bacillus as well as potential pathogens such as Vibrio and Piscirickettsia. Finally, the treatment increased survival in fish challenged with P. salmonis by more than 60%. These results demonstrate that the compound is capable of protecting fish against P. salmonis, probably by modulating the immune system and the composition of the intestinal microbiota.

Immunomodulation Evidence of Nanostructured Recombinant Proteins in Salmonid Cells.

Recent studies have demonstrated that immune-related recombinant proteins can enhance immune function, increasing host survival against infectious diseases in salmonids. This research evaluated inclusion bodies (IBs) of antimicrobial peptides (CAMPIB and HAMPIB) and a cytokine (IL1ßIB and TNFαIB) as potential immunostimulants in farmed salmonids. For this purpose, we produced five IBs (including iRFPIB as a control), and we evaluated their ability to modulate immune marker gene expression of three IBs in the RTS11 cell line by RT-qPCR. Additionally, we characterized the scale-up of IBs production by comparing two different scale systems. The results showed that CAMPIB can increase the upregulation of tnfα, il1ß, il8, and il10, HAMPIB significantly increases the upregulation of tnfα, inos, and il10, and IL1ßIB significantly upregulated the expression of tnfα, il1ß, and cox2. A comparison of IL1ßIB production showed that the yield was greater in shake flasks than in bioreactors (39 ± 1.15 mg/L and 14.5 ± 4.08 mg/L), and larger nanoparticles were produced in shake flasks (540 ± 129 nm and 427 ± 134 nm, p < 0.0001, respectively). However, compared with its shake flask counterpart, the IL1ßIB produced in a bioreactor has an increased immunomodulatory ability. Further studies are needed to understand the immune response pathways activated by IBs and the optimal production conditions in bioreactors, such as a defined medium, fed-batch production, and mechanical bacterial lysis, to increase yield.

Transcriptome analyses reveal key roles of alternative splicing regulation in atlantic salmon during the infectious process of Piscirickettsiosis disease.

In the Chilean salmon farming industry, infection by Piscirickettsia salmonis is the primary cause of the main bacterial disease known as Piscirickettsiosis, which has an overwhelming economic impact. Although it has been demonstrated that Piscirickettsiosis modifies the expression of numerous salmonids genes, it is yet unknown how alternative splicing (AS) contributes to salmonids bacterial infection. AS, has the potential to create heterogeneity at the protein and RNA levels and has been associated as a relevant molecular mechanism in the immune response of eukaryotes to several diseases. In this study, we used RNA data to survey P. salmonis-induced modifications in the AS of Atlantic salmon and found that P. salmonis infection promoted a substantial number (158,668) of AS events. Differentially spliced genes (DSG) sensitive to Piscirickettsiosis were predominantly enriched in genes involved in RNA processing, splicing and spliceosome processes (e.g., hnRNPm, hnRPc, SRSF7, SRSF45), whereas among the DSG of resistant and susceptible to Piscirickettsiosis, several metabolic and immune processes were found, most notably associated to the regulation of GTPase, lysosome and telomere organization-maintenance. Furthermore, we found that DSG were mostly not differentially expressed (5-7 %) and were implicated in distinct biological pathways. Therefore, our results underpin AS achieving a significant regulatory performance in the response of salmonids to Piscirickettsiosis.

Revealing the Salmo salar NLRP3 Inflammasome: Insights from Structural Modeling and Transcriptome Analysis.

The NLRP3, one of the most heavily studied inflammasome-related proteins in mammals, remains inadequately characterized in Atlantic salmon (Salmo salar), despite the significant commercial importance of this salmonid. The NLRP3 inflammasome is composed of the NLRP3 protein, which is associated with procaspase-1 via an adapter molecule known as ASC. This work aims to characterize the Salmo salar NLRP3 inflammasome through in silico structural modeling, functional transcript expression determination in the SHK-1 cell line in vitro, and a transcriptome analysis on Atlantic salmon. The molecular docking results suggested a similar arrangement of the ternary complex between NLRP3, ASC, and caspase-1 in both the Atlantic salmon and the mammalian NLRP3 inflammasomes. Moreover, the expression results confirmed the functionality of the SsNLRP3 inflammasome in the SHK-1 cells, as evidenced by the lipopolysaccharide-induced increase in the transcription of genes involved in inflammasome activation, including ASC and NLRP3. Additionally, the transcriptome results revealed that most of the inflammasome-related genes, including ASC, NLRP3, and caspase-1, were down-regulated in the Atlantic salmon following its adaptation to seawater (also known as parr-smolt transformation). This is correlated with a temporary detrimental effected on the immune system. Collectively, these findings offer novel insights into the evolutionarily conserved role of NLRP3.

Tenacibaculum bernardetii sp. nov., isolated from Atlantic salmon (Salmo salar L.) cultured in Chile.

Strain PVT-9aT, a novel Gram-stain-negative, aerobic, non-spore-forming, motile-by-gliding and rod-shaped bacterium, was isolated from a skin lesion of Atlantic salmon (Salmo salar L.) during a tenacibaculosis outbreak that occurred in 2016 at a Chilean fish farm. Phylogenetic analysis based on 16S rRNA gene sequencing confirmed that strain PVT-9aT belonged to the genus Tenacibaculum, being related to the closest type strains Tenacibaculum haliotis KCTC 52419T (98.49 % sequence similarity), Tenacibaculum aestuariivivum JDTF-79T (97.36 %), Tenacibaculum insulae JDTF-31T (97.29 %) and Tenacibaculum ovolyticum IFO 15947T (97.15 %). The genome size of strain PVT-9aT was 2.73 Mb with a DNA G+C content 31.09 mol%. Average nucleotide identity analysis among 30 Tenacibaculum species rendered the most similar strains as follows: T. haliotis KCTC 52419T (87.91 %), T. ovolyticum IFO 15947T (82.47 %), Tenacibaculum dicentrarchi 35/09T (81.08 %), Tenacibaculum finnmarkense gv finnmarkense TNO006T (80.91 %) and T. finnmarkense gv ulcerans TNO010T (80.96 %). Menaquinone MK-6 was the predominant respiratory quinone. The predominant cell fatty acids (>10 %) were iso-C15 : 0, iso-C15 : 1 G and iso-C15 : 0 3-OH. Phenotypic, chemotaxonomic and genomic data supported the assignment of strain PVT-9aT (=DSM 115155T=RGM 3472T) as representing a novel species of Tenacibaculum, for which the name Tenacibaculum bernardetii sp. nov. is proposed.

Heritability of Immunity Traits and Resistance of Atlantic Salmon against the Sea Louse Caligus rogercresseyi.

The immune response of Atlantic salmon to sea lice has been extensively studied, but we still do not know the mechanisms by which some fish become resistant and others do not. In this study, we estimated the heritabilities of three key proteins associated with the innate immunity and resistance of Salmo salar against the sea louse Caligus rogercresseyi. In particular, we quantified the abundance of 2 pro-inflammatory cytokines, Tnfα and Il-8, and an antioxidant enzyme, Nkef, in Atlantic salmon skin and gill tissue from 21 families and 268 individuals by indirect ELISA. This covers a wide parasite load range from low or resistant (mean sea lice ± SE = 8.7 ± 0.9) to high or susceptible (mean sea lice ± SE = 43.3 ± 2.0). Our results showed that susceptible fish had higher levels of Nkef and Tnfα than resistant fish in their gills and skin, although gill Il-8 was higher in resistant fish, while no significant differences were found in the skin. Furthermore, moderate to very high heritable genetic variation was estimated for Nkef (h2 skin: 0.96 ± 0.14 and gills: 0.97 ± 0.11) and Tnfα (h2 skin: 0.53 ± 0.17 and gills: 0.32 ± 0.14), but not for Il-8 (h2 skin: 0.22 ± 0.12 ns and gills: 0.09 ± 0.08 ns). This work provides evidence that Nkef and Tnfα protein expressions are highly heritable and related to resistance against sea lice in Atlantic salmon.

Analysis of microbiota-host communication mediated by butyrate in Atlantic salmon.

Butyrate is a microbiota-produced metabolite, sensed by host short-chain fatty acid receptors FFAR2 (Gpr43), FFAR3 (Gpr41), HCAR2 (Gpr109A), and Histone deacetylase (HDAC) that promotes microbiota-host crosstalk. Butyrate influences energy uptake, developmental and immune response in mammals. This microbial metabolite is produced by around 79 anaerobic genera present in the mammalian gut, yet little is known about the role of butyrate in the host-microbiota interaction in salmonid fish. To further our knowledge of this interaction, we analyzed the intestinal microbiota and genome of Atlantic salmon (Salmo salar), searching for butyrate-producing genera and host butyrate receptors. We identified Firmicutes, Proteobacteria, and Actinobacteria as the main butyrate-producing bacteria in the salmon gut microbiota. In the Atlantic salmon genome, we identified an expansion of genes orthologous to FFAR2 and HCAR2 receptors, and class I and IIa HDACs that are sensitive to butyrate. In addition, we determined the expression levels of orthologous of HCAR2 in the gut, spleen, and head-kidney, and FFAR2 in RTgutGC cells. The effect of butyrate on the Atlantic salmon immune response was evaluated by analyzing the pro and anti-inflammatory cytokines response in vitro in SHK-1 cells by RT-qPCR. Butyrate decreased the expression of the pro-inflammatory cytokine IL-1ß and increased anti-inflammatory IL-10 and TGF-ß cytokines. Butyrate also reduced the expression of interferon-alpha, Mx, and PKR, and decreased the viral load at a higher concentration (4 mM) in cells treated with this molecule before the infection with Infectious Pancreatic Necrosis Virus (IPNV) by mechanisms independent of FFAR2, FFAR3 and HCAR2 expression that probably inhibit HDAC. Moreover, butyrate modified phosphorylation of cytoplasmic proteins in RTgutGC cells. Our data allow us to infer that Atlantic salmon have the ability to sense butyrate produced by their gut microbiota via different specific targets, through which butyrate modulates the immune response of pro and anti-inflammatory cytokines and the antiviral response.

Live and inactivated Piscirickettsia salmonis activated nutritional immunity in Atlantic salmon (Salmo salar).

Nutritional immunity regulates the homeostasis of micronutrients such as iron, manganese, and zinc at the systemic and cellular levels, preventing the invading microorganisms from gaining access and thereby limiting their growth. Therefore, the objective of this study was to evaluate the activation of nutritional immunity in specimens of Atlantic salmon (Salmo salar) that are intraperitoneally stimulated with both live and inactivated Piscirickettsia salmonis. The study used liver tissue and blood/plasma samples on days 3, 7, and 14 post-injections (dpi) for the analysis. Genetic material (DNA) of P. salmonis was detected in the liver tissue of fish stimulated with both live and inactivated P. salmonis at 14 dpi. Additionally, the hematocrit percentage decreased at 3 and 7 dpi in fish stimulated with live P. salmonis, unchanged in fish challenged with inactivated P. salmonis. On the other hand, plasma iron content decreased during the experimental course in fish stimulated with both live and inactivated P. salmonis, although this decrease was statistically significant only at 3 dpi. Regarding the immune-nutritional markers such as tfr1, dmt1, and ireg1 were modulated in the two experimental conditions, compared to zip8, ft-h, and hamp, which were down-regulated in fish stimulated with live and inactivated P. salmonis during the course experimental. Finally, the intracellular iron content in the liver increased at 7 and 14 dpi in fish stimulated with live and inactivated P. salmonis, while the zinc content decreased at 14 dpi under both experimental conditions. However, stimulation with live and inactivated P. salmonis did not alter the manganese content in the fish. The results suggest that nutritional immunity does not distinguish between live and inactivated P. salmonis and elicits a similar immune response. Probably, this immune mechanism would be self-activated with the detection of PAMPs, instead of a sequestration and/or competition of micronutrients by the living microorganism.

Healthy and infected Atlantic salmon (Salmo salar) skin-mucus response to Tenacibaculum dicentrarchi under in vitro conditions.

Tenacibaculosis caused by Tenacibaculum dicentrarchi is the second most important bacterial disease that affects the Chilean salmon industry. The impacted fish show severe external gross skin lesions on different areas of the body. The external mucus layer that covers fish skin contains numerous immune substances that act as one of the main defense barriers against microbial colonization and invasions by potential pathogens. The present in vitro study aimed to evaluate and elucidate the role of the external mucus layer in the susceptibility of Atlantic salmon (Salmo salar) to three Chilean T. dicentrarchi strains and the type strain. For this, mucus collected from healthy and diseased (i.e., with T. dicentrarchi) Atlantic salmon were used, and various antibacterial and inflammatory parameters were analysed. The T. dicentrarchi strains were attracted to the mucus of Atlantic salmon regardless of health status. All four strains adhered to the skin mucus and very quickly grew using the mucus nutrients. Once infection was established, different mucosal defense components were activated in the fish, but the levels of bactericidal activity and of other enzymes were insufficient to eliminate T. dicentrarchi. Alternatively, this pathogen may be able to neutralize or evade these mechanisms. Therefore, the survival of T. dicentrarchi in fish skin mucus could be relevant to facilitate the colonization and subsequent invasion of hosts. The given in vitro results suggest that greater attention should be given to fish skin mucus as a primary defense against T. dicentrarchi.

Co-Infection by LF-89-Like and EM-90-Like Genogroups of Piscirickettsia Salmonis in Farmed Atlantic Salmon in Chile: Implications for Surveillance and Control of Piscirickettsiosis.

Piscirickettsiosis (SRS), caused by Piscirickettsia salmonis, is the main infectious disease that affects farmed Atlantic salmon in Chile. Currently, the official surveillance and control plan for SRS in Chile is based only on the detection of P. salmonis, but neither of its genogroups (LF-89-like and EM-90-like) are included. Surveillance at the genogroup level is essential not only for defining and evaluating the vaccination strategy against SRS, but it is also of utmost importance for early diagnosis, clinical prognosis in the field, treatment, and control of the disease. The objectives of this study were to characterize the spatio-temporal distribution of P. salmonis genogroups using genogroup-specific real-time probe-based polymerase chain reaction (qPCR) to discriminate between LF-89-like and EM-90-like within and between seawater farms, individual fish, and tissues/organs during early infection in Atlantic salmon under field conditions. The spatio-temporal distribution of LF-89-like and EM-90-like was shown to be highly variable within and between seawater farms. P. salmonis infection was also proven to be caused by both genogroups at farm, fish, and tissue levels. Our study demonstrated for the first time a complex co-infection by P. salmonis LF-89-like and EM-90-like in Atlantic salmon. Liver nodules (moderate and severe) were strongly associated with EM-90-like infection, but this phenotype was not detected by infection with LF-89-like or co-infection of both genogroups. The detection rate of P. salmonis LF-89-like increased significantly between 2017 and 2021 and was the most prevalent genogroup in Chilean salmon aquaculture during this period. Lastly, a novel strategy to identify P. salmonis genogroups based on novel genogroup-specific qPCR for LF-89-like and EM-90-like genogroups is suggested.

Unveiling the Role of Dynamic Alternative Splicing Modulation After Infestation with Sea Lice (Caligus rogercresseyi) in Atlantic Salmon.

Sea lice are pathogenic marine ectoparasite copepods that represent a severe risk to the worldwide salmon industry. Several transcriptomic investigations have characterized the regulation of gene expression response of Atlantic salmon to sea lice infestation. These studies have focused on the levels of transcript, overlooking the potentially relevant role of alternative splicing (AS), which corresponds to an essential control mechanism of gene expression through RNA processing. In the present study, we performed a genome-wide bioinformatics characterization of differential AS event dynamics in control and infested C. rogercresseyi Atlantic salmon and in resistant and susceptible phenotypes. We identified a significant rise of alternative splicing events and AS genes after infestation and 176 differential alternative splicing events (DASE) from 133 genes. In addition, a higher number of DASE and AS genes were observed among resistant and susceptible phenotypes. Functional annotation of AS genes shows several terms and pathways associated with behavior, RNA splicing, immune response, and RNA binding. Furthermore, three protein-coding genes were identified undergoing differential transcript usage events, among resistant and susceptible phenotypes. Our findings support AS performing a relevant regulatory role in the response of salmonids to sea lice infestation.

KLF17 is an important regulatory component of the transcriptomic response of Atlantic salmon macrophages to Piscirickettsia salmonis infection.

Piscirickettsia salmonis is the most important health problem facing Chilean Aquaculture. Previous reports suggest that P. salmonis can survive in salmonid macrophages by interfering with the host immune response. However, the relevant aspects of the molecular pathogenesis of P. salmonis have been poorly characterized. In this work, we evaluated the transcriptomic changes in macrophage-like cell line SHK-1 infected with P. salmonis at 24- and 48-hours post-infection (hpi) and generated network models of the macrophage response to the infection using co-expression analysis and regulatory transcription factor-target gene information. Transcriptomic analysis showed that 635 genes were differentially expressed after 24- and/or 48-hpi. The pattern of expression of these genes was analyzed by weighted co-expression network analysis (WGCNA), which classified genes into 4 modules of expression, comprising early responses to the bacterium. Induced genes included genes involved in metabolism and cell differentiation, intracellular transportation, and cytoskeleton reorganization, while repressed genes included genes involved in extracellular matrix organization and RNA metabolism. To understand how these expression changes are orchestrated and to pinpoint relevant transcription factors (TFs) controlling the response, we established a curated database of TF-target gene regulatory interactions in Salmo salar, SalSaDB. Using this resource, together with co-expression module data, we generated infection context-specific networks that were analyzed to determine highly connected TF nodes. We found that the most connected TF of the 24- and 48-hpi response networks is KLF17, an ortholog of the KLF4 TF involved in the polarization of macrophages to an M2-phenotype in mammals. Interestingly, while KLF17 is induced by P. salmonis infection, other TFs, such as NOTCH3 and NFATC1, whose orthologs in mammals are related to M1-like macrophages, are repressed. In sum, our results suggest the induction of early regulatory events associated with an M2-like phenotype of macrophages that drives effectors related to the lysosome, RNA metabolism, cytoskeleton organization, and extracellular matrix remodeling. Moreover, the M1-like response seems delayed in generating an effective response, suggesting a polarization towards M2-like macrophages that allows the survival of P. salmonis. This work also contributes to SalSaDB, a curated database of TF-target gene interactions that is freely available for the Atlantic salmon community.

Isolation of a New Infectious Pancreatic Necrosis Virus (IPNV) Variant from Genetically Resistant Farmed Atlantic Salmon (Salmo salar) during 2021-2022.

Infectious pancreatic necrosis (IPN), caused by IPNV, affects several species of farmed fish, particularly Atlantic salmon, and is responsible for significant economic losses in salmon aquaculture globally. Despite the introduction of genetically resistant farmed Atlantic salmon and vaccination strategies in the Chilean salmon industry since 2019, the number of IPN outbreaks has been increasing in farmed Atlantic salmon in the freshwater phase. This study examined gross and histopathological lesions of IPNV-affected fish, as well as the IPNV nucleotide sequence encoding the VP2 protein in clinical cases. The mortality reached 0.4% per day, and the cumulative mortality was from 0.4 to 3.5%. IPNV was isolated in the CHSE-214 cell line and was confirmed by RT-PCR, and VP2 sequence analysis. The analyzed viruses belong to IPNV genotype 5 and have 11 mutations in their VP2 protein. This is the first report of IPN outbreaks in farmed Atlantic salmon genetically resistant to IPNV in Chile. Similar outbreaks were previously reported in Scotland and Norway during 2018 and 2019, respectively. This study highlights the importance of maintaining a comprehensive surveillance program in conjunction with the use of farmed Atlantic salmon genetically resistant to IPNV.

Reference Intervals for Blood Biomarkers in Farmed Atlantic Salmon, Coho Salmon and Rainbow Trout in Chile: Promoting a Preventive Approach in Aquamedicine.

The mission of veterinary clinical pathology is to support the diagnostic process by using tests to measure different blood biomarkers to support decision making about farmed fish health and welfare. The objective of this study is to provide reference intervals (RIs) for 44 key hematological, blood biochemistry, blood gasometry and hormones biomarkers for the three most economically important farmed salmonid species in Chile (Atlantic salmon, coho salmon and rainbow trout) during the freshwater (presmolt and smolt age range) and seawater stages (post-smolt and adult age range). Our results confirmed that the concentration or activity of most blood biomarkers depend on the salmonid species, age range and/or the interaction between them, and they are often biologically related to each other. Erythogram and leukogram profiles revealed a similar distribution in rainbow trout and coho salmon, but those in Atlantic salmon were significantly different. While the activity of the most clinically important plasma enzymes demonstrated a similar profile in Atlantic salmon and rainbow trout, coho salmon demonstrated a significantly different distribution. Plasma electrolyte and mineral profiles showed significant differences between salmonid species, especially for rainbow trout, while Atlantic salmon and coho salmon demonstrated a high degree of similarity. Furthermore, electrolytes, minerals and blood gasometry biomarkers were significantly different between age ranges, suggesting a considerably different distribution between freshwater and seawater-farmed fish. The RIs of clinically healthy fish described in this study take into account the high biological variation of farmed fish in Chile, as the 3.059 individuals came from 78 different fish farms, both freshwater and seawater, and blood samples were collected using the same pre-analytical protocol. Likewise, our study provides the Chilean salmon farming industry with standardized protocols that can be used routinely and provides valuable information to improve the preventive vision of aquamedicine through the application of blood biomarkers to support and optimize health, welfare and husbandry management in the salmon farming industry.

Physiological Response of Atlantic Salmon (Salmo salar) to Long-Term Exposure to an Anesthetic Obtained from Heterosigma akashiwo.

Despite the invaluable role of anesthetics as a tool for ensuring animal welfare in stressful situations, there is currently a lack of anesthetic drugs that meet the requirements of intensive aquaculture. In response to the growing interest in anesthetic substances of natural origin, this study evaluated the physiological and health impact of an anesthetic based on an extract of the microalga Heterosigma akashiwo on juvenile salmon (Salmo salar) exposed for a period of 72 h. To simulate a condition closer to reality where fish are subjected to stimuli (e.g., transport), the animals were exposed to 50 mg L-1 of algal extract and to physical stress. Functional, physiological, and histological parameters were evaluated in blood and tissues at different sampling periods (0, 24, and 72 h). There was no mortality and the induction and recovery times observed were within the established criteria for anesthetic efficacy. The anesthetic extract did not induce any side effects, such as stress or metabolic damage, indicating that this extract is a viable option for supporting fish welfare during deleterious events. This study provides information to support that the anesthetic extract tested, derived from H. akashiwo, is a promising candidate drug for operations requiring sedation (e.g., Salmonid transport).