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Salmonid rickettsial septicemia (SRS), caused by Piscirickettsia salmonis , is a major challenge in Chilean aquaculture. We evaluated the impact of the vaccine on fish survival, bacterial load, and iron metabolism-related gene expression under field conditions. Atlantic salmon received either a pentavalent inactivated vaccine plus a live attenuated P. salmonis vaccine (SIA) or a tetravalent control vaccine (SS). While survival was similar early (≤ 28 weeks post-seawater transfer), SIA-vaccinated fish showed greater survival by week 41 (85% vs. 52%). Quantitative PCR confirmed a reduced bacterial load in the SIA group during active infection. Expression analysis revealed distinct temporal regulation of iron metabolism genes: ferritin and hepcidin were upregulated in freshwater, whereas transferrin and its receptor were elevated in seawater in the SIA group, suggesting a link between iron homeostasis and vaccine-induced protection. To further investigate the role of transferrin, we generated transferrin-knockout (TF-KO) phagocytes using CRISPR/Cas9. In vitro infection assays revealed that, compared with wild-type (TF-WT) cells, TF-KO cells presented reduced cytopathic effects, decreased formation of P. salmonis -containing vacuoles (PCVs), and improved viability. Surprisingly, no differences in bacterial load or iron-related gene expression were detected between TF-KO and TF-WT cells, indicating that transferrin disruption did not directly alter iron homeostasis. Global transcriptomic analysis revealed 311 differentially expressed genes in TF-KO cells, with functional enrichment in metal-binding and zinc-dependent processes but no direct association with iron metabolism. These findings suggest that transferrin deficiency confers an infection-tolerant phenotype through transcriptional reprogramming unrelated to iron regulation, highlighting novel mechanisms of host‒pathogen interactions and potential avenues for SRS control in aquaculture.

In Chile, Piscirickettsia salmonis contains two genetically isolated genogroups, LF-89 and EM-90. However, the impact of a potential co-infection with these two variants on Salmonid Rickettsial Septicemia (SRS) in Atlantic salmon ( Salmo salar ) remains largely unexplored. In our study, we evaluated the effect of P. salmonis LF-89-like and EM-90-like co-infection on post-smolt Atlantic salmon after an intraperitoneal challenge to compare changes in disease dynamics and host immune response. Co-infected fish had a significantly lower survival rate (24.1%) at 21 days post-challenge (dpc), compared with EM-90-like single-infected fish (40.3%). In contrast, all the LF-89-like single-infected fish survived. In addition, co-infected fish presented a higher presence of clinical lesions than any of the single-infected fish. The gene expression of salmon immune-related biomarkers evaluated in the head kidney, spleen, and liver showed that the EM-90-like isolate and the co-infection induced the up-regulation of cytokines (e.g., il-1β , ifnγ , il8 , il10 ), antimicrobial peptides ( hepdicin ) and pattern recognition receptors (PRRs), such as TLR5s . Furthermore, in serum samples from EM-90-like and co-infected fish, an increase in the total IgM level was observed. Interestingly, specific IgM against P. salmonis showed greater detection of EM-90-like antigens in LF-89-like infected fish serum (cross-reaction). These data provide evidence that P. salmonis LF-89-like and EM-90-like interactions can modulate SRS disease dynamics in Atlantic salmon, causing a synergistic effect that increases the severity of the disease and the mortality rate of the fish. Overall, this study contributes to achieving a better understanding of P. salmonis population dynamics.

Piscirickettsia salmonis is the pathogen that has most affected the Chilean salmon industry for over 30 years. Considering the problems of excessive use of antibiotics, it is necessary to find new strategies to control this pathogen. Antivirulence therapy is an alternative to reduce the virulence of pathogens without affecting their growth. Polyphenolic compounds have been studied for their antiviral capacity. In this study, the capacity of quercetin and silybin to reduce the intracellular replication of P. salmonis in SHK-1 cells was evaluated. For this, three different infection protocols in Salmon Head Kidney-1(SHK-1) cells were used: co-incubation for 24 h, pre-incubation for 24 h prior to infection, and post-incubation for 24 h after infection. In addition, the effect of co-incubation in rainbow trout intestinal epithelial cells (RTgutGC) and the effect on the phagocytic capacity of SHK-1 cells were evaluated. The results obtained showed that quercetin and silybin decreased the intracellular replication of P. salmonis in SHK-1 cells when they were co-incubated for 24 h; however, they did not have the same effect in RTgutGC cells. On the other hand, both compounds decreased the phagocytosis of SHK-1 cells during co-incubation. These results are promising for the study of new treatments against P. salmonis.

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.

is a globally distributed aquatic bacterium and a component of the normal salmon microbiome. It has significant biological and economic impact on Chilean salmon aquaculture due to the highly fatal disease, piscirickettsiosis. Unsuccessful attempts to prevent and treat this disease have resulted in heavy use of antimicrobials with adverse effects on the aquatic environment and piscine and human health. Evidence suggests could be a bacterium with relative pathogenic potential on farmed salmonids and other fishes that triggers piscirickettsiosis under particular conditions in the salmon and its environment. Application of a damage-response framework analysis could define the steps from asymptomatic infection to symptomatic disease, help tailor improved approaches to disease prevention and management, and, in turn, help avoid heavy use of antimicrobials which have global effects on animal health, human health, and environmental biodiversity (the One Health concept).

The management of bacterial pathogens remains a key challenge of aquaculture. The marine gammaproteobacterium Piscirickettsia salmonis is the etiological agent of piscirickettsiosis and causes multi-systemic infections in different salmon species, resulting in considerable mortality and substantial commercial losses. Here, we elucidate its global diversity, evolution, and selection during human interventions. Our comprehensive analysis of 73 closed, high quality genome sequences covered strains from major outbreaks and was supplemented by an analysis of all P. salmonis 16S rRNA gene sequences and metagenomic reads available in public databases. Genome comparison showed that Piscirickettsia comprises at least three distinct, genetically isolated species of which two showed evidence for continuing speciation. However, at least twice the number of species exist in marine fish or seawater. A hallmark of Piscirickettsia diversification is the unprecedented amount and diversity of transposases which are particularly active in subgroups undergoing rapid speciation and are key to the acquisition of novel genes and to pseudogenization. Several group-specific genes are involved in surface antigen synthesis and may explain the differences in virulence between strains. However, the frequent failure of antibiotic treatment of piscirickettsiosis outbreaks cannot be explained by horizontal acquisition of resistance genes which so far occurred only very rarely. Besides revealing a dynamic diversification of an important pathogen, our study also provides the data for improving its surveillance, predicting the emergence of novel lineages, and adapting aquaculture management, and thereby contributes towards the sustainability of salmon farming.

Vaccination is a widely used control strategy to prevent Piscirickettsia salmonis causing disease in salmon farming. However, it is not known why all the currently available commercial vaccines generally fail to protect against this pathogenic bacteria. Here, we report, from two different populations, that between-family variation is a strong intrinsic factor that determines vaccine protection for this disease. While in some full-sib families, the protection added by vaccination increased the survival time in 13 days in comparison with their unvaccinated siblings; in other families, there was no added protection by vaccination or even it was slightly negative. Resistance to P. salmonis, measured as days to death, was higher in vaccinated than unvaccinated fish, but only a moderate positive genetic correlation was obtained between these traits. This disputes a previous hypothesis, that stated that both traits were fully controlled by the same genes, and challenges the use of unvaccinated fish as gold standard for evaluating and selecting fish resistant to P. salmonis , particularly if the offspring will be vaccinated. More studies are necessary to evaluate if variation in the host immune response to vaccination could explain the between-family differences in resistance observed in vaccinated fish.

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(NN ) ]ClO where NN = 6-((quinolin-2-ylmethylene)amino)-2H-chromen-2-one. The antibacterial capacity of [Cu(NN ) ]ClO was determined. Subsequently, the effect of the administration of [Cu(NN ) ]ClO on the growth of , modulation of the immune system and the intestinal microbiota was studied. Finally, the ability to protect against a challenge with 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-1β, 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 and as well as potential pathogens such as and . Finally, the treatment increased survival in fish challenged with by more than 60%. These results demonstrate that the compound is capable of protecting fish against , probably by modulating the immune system and the composition of the intestinal microbiota.

Electrochemical detection of solid-phase isothermal recombinase polymerase amplification (RPA) of Piscirickettsia salmonis in salmon genomic DNA is reported. The electrochemical biosensor was constructed by surface functionalization of gold electrodes with a thiolated forward primer specific to the genomic region of interest. Solid-phase RPA and primer elongation were achieved in the presence of the specific target sequence and biotinylated reverse primers. The formation of the subsequent surface-tethered duplex amplicons was electrochemically monitored via addition of streptavidin-linked HRP upon completion of solid-phase RPA. Successful quantitative amplification and detection were achieved in less than 1 h at 37 °C, calibrating with PCR-amplified genomic DNA standards and achieving a limit of detection of 5 · 10 −8  μg ml −1 (3 · 10 3 copies in 10 μl). The presented system was applied to the analysis of eight real salmon samples, and the method was also compared to qPCR analysis, observing an excellent degree of correlation. Graphical abstract Schematic of use of electrochemical RPA for detection of Psiricketessia salmonis in salmon liver

Type II interferon gamma (IFNγ) is a pleiotropic cytokine capable of modulating the innate and adaptive immune responses which has been widely characterized in several teleost families. In fish, IFNγ stimulates the expression of cytokines and chemokines associated with the pro-inflammatory response and enhances the production of nitrogen and oxygen reactive species in phagocytic cells. This work studied the effect of IFNγ on the expression of cell-surface markers on splenocytes of Atlantic salmon ( Salmo salar ). In vitro results showed that subpopulations of mononuclear splenocytes cultured for 15 days were capable of increasing gene expression and protein availability of cell-surface markers such as CD80/86, CD83 and MHC II, after being stimulated with recombinant IFNγ. These results were observed for subpopulations with characteristics associated with monocytes (51%), and features that could be related to lymphocytes (46.3%). In addition, a decrease in the expression of zbtb46 was detected in IFNγ-stimulated splenocytes. Finally, the expression of IFNγ and cell-surface markers was assessed in Atlantic salmon under field conditions. In vivo results showed that the expression of ifnγ increased simultaneously with the up-regulation of cd80/86 , cd83 and mhcii during a natural outbreak of Piscirickettsia salmonis . Overall, the results obtained in this study allow us to propose IFNγ as a candidate molecule to stimulate the phenotypic progression of a small population of immune cells, which will increase antigen presenting cells markers. Thereby, modulatory strategies using IFNγ may generate a robust and coordinated immune response in fish against pathogens that affect aquaculture.