Search Results (15)

Aquaculture is currently the fastest-growing sector in animal production, with an average annual growth rate of 7.5% since 1970. In Chile, the industry is largely driven by salmonid farming, with Salmo salar (Atlantic salmon) accounting for over 65% of national production. This species has shown the most significant growth within the sector. This growth is achieved by having high-density farming, which results in high levels of stress due to overcrowding and the appearance of pathogens such as the Infectious Salmon Anemia (ISA) virus, Bacterial Kidney Disease (BKD), Caligus sea lice (Caligus rogercresseyi), and Piscirickettsiosis (SRS) caused by Piscirickettsia salmonis, among others. This study evaluated the toxicity of the two most commonly used antibiotics in the salmon industry—oxytetracycline and florfenicol—at four concentrations each, using primary liver cell cultures of Atlantic salmon (Salmo salar). Oxidative stress was assessed through enzymatic activity and gene expression of oxidative markers, including cytochrome P450, catalase (CAT), superoxide dismutase (SOD), glutathione reductase (GR), and glutathione peroxidase (GPx). Samples were analyzed at 1, 3, 6, 12, and 48 h post-exposure. These findings reveal time- and dose-dependent oxidative responses in salmon liver cells to OTC and FLO, providing critical insights into the sublethal cellular effects of antibiotics commonly used in aquaculture, which indicates the presence of a high amount of free radicals in the liver cells, indicating toxicity of both antibiotics. Full article

Effective control of bacterial infections remains a significant challenge in aquaculture. The marine bacterium Piscirickettsia salmonis (P. salmonis), responsible for piscirickettsiosis, causes widespread infections in various salmon species, leading to substantial mortality and economic losses. Despite efforts to genetically characterize P. salmonis, critical gaps persist in understanding its virulence factors, antimicrobial resistance genes, and single-nucleotide polymorphisms (SNPs). This study addresses these gaps through a comparative analysis of the pan-genome and core genomes of 80 P. salmonis strains from different geographical regions and genogroups. P. salmonis had an open pan-genome consisting of 14,564 genes, with a core genome of 1257 conserved genes. Eleven virulence-related genes were identified in the pan-genome, categorized into five functional groups, providing new insights into the pathogenicity of P. salmonis. Unique SNPs were detected in four key genes (gyrA, dnaK, rpoB, and ftsZ), serving as robust molecular markers for distinguishing the LF and EM genogroups. Notably, AMR genes identified in four LF strains suggest evolutionary adaptations under selective pressure. Functional annotation of the core genomes using the gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases demonstrated conserved gene clusters linked to essential intracellular survival mechanisms and bacterial pathogenicity. These findings suggest a direct association between core genome features and variations in pathogenesis and host–pathogen interactions across genogroups. Phylogenetic reconstruction further highlighted the influence of AMR genes on strain divergence. Collectively, this study enhances the genomic understanding of P. salmonis and lays the groundwork for improved diagnostic tools and targeted therapeutics to manage piscirickettsiosis in aquaculture. Full article

Sustainably farmed Atlantic salmon could drive global food system solutions by contributing essential nutrients to the human diet while delivering high-quality protein. One of the biggest obstacles to sustainable salmon aquaculture in Chile is the prevalence of piscirickettsiosis disease caused by the Gram-negative bacteria Piscirickettsia salmonis and the excessive amount of antibiotics used to eradicate this disease. Farmed salmon products can be consumed without prior processing and therefore present a substantial risk for the transfer of resistant pathogens to humans. Antibiotics also carry the risk of antibiotic residues and damage to the environment. An alternative to antibiotics is the use of natural antimicrobials without the negative influence on the consumer’s microbiome. Here, we evaluate the potential antimicrobial activity against P. salmonis of the marine microalgae Microchloropsis gaditana. A non-genetically modified M. gaditana was grown with nitrogen deprivation to improve the synthesis of the eicosapentaenoic fatty acid (EPA). A spray-dried M. gaditana concentrate (Mg) was elaborated and given to Atlantic salmon for a period of 49 days, and serum and fillet samples were collected. Our results showed a significant increase in the nutritional quality improving the levels of EPA+ Docosapentaenoic acid (DPA) (23%) and Vitamin D₃ (106%) of the fillets treated with Mg. Fish fed serum were challenged with P. salmonis, and serum antibacterial activity was measured. Sera from fish fed Mg-enriched diets showed a significant increase in antibacterial activity (85.68%) against P. salmonis. Our results indicate that Mg can be used as a viable alternative to address the critical problem of microbial resistance and to assure consumers that farm-raised Atlantic salmon is safe. Full article

Salmonid rickettsial septicemia (SRS) is a critical sanitary problem in the Chilean aquaculture industry since it induces the highest mortality rate in salmonids among all infectious diseases. Piscirickettsia salmonis, a facultative intracellular bacterium, is the biological agent of SRS. In Chile, two genogroups of P. salmonis, designated as LF-89 and EM-90, have been identified. Previous studies suggested that their cohabitation triggers the expression of virulence effectors, which may be related to a higher pathogenicity in salmonids during co-infection with both P. salmonis genogroups. Therefore, we aimed to evaluate if the physical contact between two isolates from LF-89 and EM-90 is necessary to activate this effect. Through a spatially separated in vivo co-culture inside Atlantic salmon (Salmo salar) post smolts and RNA-seq analysis, we compared the differentially expressed genes (DEGs) with previous results from an in vivo mixed co-culture. The results showed that although the LF-89-like isolate and the EM-90-like isolate had a similar DEG profile under both co-culture conditions, important virulence factors observed during the mixed co-cultures (i.e., flagellar-related genes, CydD, and NCS2) were absent in the spatially separated co-cultures. Hence, the synergistic effect linked to increased pathogenicity to the host may be driven by the physical co-localization and contact between the P. salmonis LF-89-like and EM-90-like isolates. Full article

For the last 30 years, Piscirickettsia salmonis has caused major economic losses to the aquaculture industry as the aetiological agent for the piscirickettsiosis disease. Replacing the current interventions, based on antibiotics, with natural alternatives (e.g., organic acids) represents a priority. With this study, we aimed to better understand their biological mechanism of action in an in vitro model of infection with salmon epithelial cells (CHSE-214). Our first observation revealed that at the sub-inhibitory concentration of 0.5%, the organic acid blend (Aq) protected epithelial cell integrity and significantly reduced P. salmonis invasion. The MIC was established at 1% Aq and the MBC at 2% against P. salmonis. The sub-inhibitory concentration significantly increased the expression of the antimicrobial peptides Cath2 and Hepcidin1, and stimulated the activity of the innate immune effector iNOS. The increase in iNOS activity also led to higher levels of nitric oxide (NO) being released in the extracellular space. The exposure of P. salmonis to the endogenous NO caused an increase in bacterial lipid peroxidation levels, a damaging effect which can ultimately reduce the pathogen’s ability to attach or multiply intracellularly. We also demonstrate that the increased NO release by the host CHSE-214 cells is a consequence of direct exposure to Aq and is not dependent on P. salmonis infection. Additionally, the presence of Aq during P. salmonis infection of CHSE-214 cells significantly mitigated the expression of the pro-inflammatory cytokines IL-1β, IL-8, IL-12, and IFNγ. Taken together, these results indicate that, unlike antibiotics, natural antimicrobials can weaponize the iNOS pathway and secreted nitric oxide to reduce infection and inflammation in a Piscirickettsia salmonis in vitro model of infection. Full article

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-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 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. Full article

Piscirickettsiosis, the main infectious disease affecting salmon farming in Chile, still has no efficient control measures. Piscirickettsia salmonis is a facultative intracellular bacterium that can survive and replicate within the host macrophages, evading the immune response. Triterpenic saponins obtained from the Quillaja saponaria tree have been widely studied, and have been shown to be immunomodulatory agents, suitable for feed and vaccine applications for veterinary and human uses. The impact of the oral administration of two extracts of Quillaja saponins on the infection of P. salmonis in Salmo salar and the corresponding gene expressions of immunomarkers were studied under three in vivo models. In the intraperitoneal challenge model, the group fed with Quillaja extracts showed lower mortality (29.1% treated vs. 37.5% control). Similar results were obtained in the cohabitation model trial (36.3% vs. 60.0%). In the commercial pilot trial, the results showed a significant reduction of 71.3% in mortality caused by P. salmonis (0.51% vs. 1.78%) and antibiotic use (reduction of 66.6% compared to untreated control). Also, Quillaja extracts significantly modulated the expression of IFN-II and CD8. These results represent evidence supporting the future use of purified Quillaja extracts as a natural non-pharmacological strategy for the prevention and control of P. salmonis infections in salmon. Full article

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. Full article

In Atlantic salmon, vaccines have failed to control and prevent Piscirickettsiosis, for reasons that remain elusive. In this study, we report the efficacy of two commercial vaccines developed with the Piscirickettsia salmonis isolates AL100005 and AL 20542 against another two genogroups which are considered highly and ubiquitously prevalent in Chile: LF-89 and EM-90. Two cohabitation trials were performed to mimic field conditions and vaccine performance: (1) post-smolt fish were challenged with a single infection of LF-89, (2) adults were coinfected with EM-90, and a low level coinfection of sea lice. In the first trial, the vaccine delayed smolt mortalities by two days; however, unvaccinated and vaccinated fish did not show significant differences in survival (unvaccinated: 60.3%, vaccinated: 56.7%; p = 0.28). In the second trial, mortality started three days later for vaccinated fish than unvaccinated fish. However, unvaccinated and vaccinated fish did not show significant differences in survival (unvaccinated: 64.6%, vaccinated: 60.2%, p = 0.58). Thus, we found no evidence that the evaluated vaccines confer effective protection against the genogroups LF-89 and EM-90 of P. salmonis with estimated relative survival proportions (RPSs) of −9% and −12%, respectively. More studies are necessary to evaluate whether pathogen heterogeneity is a key determinant of the lack of vaccine efficacy against P. salmonis. Full article

Piscirickettsia salmonis is the etiologic agent of piscirickettsiosis, a disease that causes significant losses in the salmon farming industry. In order to unveil the pathogenic mechanisms of P. salmonis, appropriate molecular and cellular studies in multiple cell lines with different origins need to be conducted. Toward that end, we established a cell viability assay that is suitable for high-throughput analysis using the alamarBlue reagent to follow the distinct stages of the bacterial infection cycle. Changes in host cell viability can be easily detected using either an absorbance- or fluorescence-based plate reader. Our method accurately tracked the infection cycle across two different Atlantic salmon-derived cell lines, with macrophage and epithelial cell properties, and zebrafish primary cell cultures. Analyses were also carried out to quantify intracellular bacterial replication in combination with fluorescence microscopy to visualize P. salmonis and cellular structures in fixed cells. In addition, dual gene expression analysis showed that the pro-inflammatory cytokines IL-6, IL-12, and TNFα were upregulated, while the cytokines IL1b and IFNγ were downregulated in the three cell culture types. The expression of the P. salmonis metal uptake and heme acquisition genes, together with the toxin and effector genes ospD3, ymt, pipB2 and pepO, were upregulated at the early and late stages of infection regardless of the cell culture type. On the other hand, Dot/Icm secretion system genes as well as stationary state and nutrient scarcity-related genes were upregulated only at the late stage of P. salmonis intracellular infection. We propose that these genes encoding putative P. salmonis virulence factors and immune-related proteins could be suitable biomarkers of P. salmonis infection. The infection protocol and cell viability assay described here provide a reliable method to compare the molecular and cellular changes induced by P. salmonis in other cell lines and has the potential to be used for high-throughput screenings of novel antimicrobials targeting this important fish intracellular pathogen. Full article

Piscirickettsia salmonis, the etiological agent of Piscirickettsiosis, is a Gram-negative and facultative intracellular pathogen that has affected the Chilean salmon industry since 1989. The bacterium is highly aggressive and can survive and replicate within fish macrophages using the Dot/Icm secretion system to evade the host’s immune response and spread systemically. To date, no efficient control measures have been developed for this disease; therefore, the producers use large amounts of antibiotics to control this pathogen. In this frame, this work has focused on evaluating the use of saponins from Quillaja saponaria as a new alternative to control the Piscirickettsiosis. It has been previously reported that purified extract of Q. saponaria (PQSE) displays both antimicrobial activity against pathogenic bacteria and viruses and adjuvant properties. Our results show that PQSE does not present antimicrobial activity against P. salmonis, although it reduces P. salmonis infection in an in vitro model, promoting the phagosome–lysosome fusion. Additionally, we demonstrate that PQSE modulates the expression of IL-12 and IL-10 in infected cells, promoting the immune response against the pathogen and reducing the expression of pathogen virulence genes. These results together strongly argue for specific anti-invasion and anti-intracellular replication effects induced by the PQSE in macrophages. Full article

Piscirickettsiasalmonis is an intracellular bacterial fish pathogen that causes piscirickettsiosis, a disease with numerous negative impacts in the Chilean salmon farming industry. Although transcriptomic studies of P. salmonis and its host have been performed, dual host–pathogen proteomic approaches during infection are still missing. Considering that gene expression does not always correspond with observed phenotype, and bacteriological culture studies inadequately reflect infection conditions, to improve the existing knowledge for the pathogenicity of P. salmonis, we present here a global proteomic profiling of Salmon salar macrophage-like cell cultures infected with P. salmonis LF-89. The proteomic analyses identified several P. salmonis proteins from two temporally different stages of macrophages infection, some of them related to key functions for bacterial survival in other intracellular pathogens. Metabolic differences were observed in early-stage infection bacteria, compared to late-stage infections. Virulence factors related to membrane, lipopolysaccharide (LPS) and surface component modifications, cell motility, toxins, and secretion systems also varied between the infection stages. Pilus proteins, beta-hemolysin, and the type VI secretion system (T6SS) were characteristic of the early-infection stage, while fimbria, upregulation of 10 toxins or effector proteins, and the Dot/Icm type IV secretion system (T4SS) were representative of the late-infection stage bacteria. Previously described virulence-related genes in P. salmonis plasmids were identified by proteomic assays during infection in SHK-1 cells, accompanied by an increase of mobile-related elements. By comparing the infected and un-infected proteome of SHK-1 cells, we observed changes in cellular and redox homeostasis; innate immune response; microtubules and actin cytoskeleton organization and dynamics; alteration in phagosome components, iron transport, and metabolism; and amino acids, nucleoside, and nucleotide metabolism, together with an overall energy and ATP production alteration. Our global proteomic profiling and the current knowledge of the P. salmonis infection process allowed us to propose a model of the macrophage–P. salmonis interaction. Full article

Piscirickettsia salmonis is the causative agent of Piscirickettsiosis, an infectious disease with a high economic impact on the Chilean salmonid aquaculture industry. This bacterium produces biofilm as a potential resistance and persistence strategy against stressful environmental stimuli. However, the in vitro culture conditions that modulate biofilm formation as well as the effect of sessile bacteria on virulence and immune gene expression in host cells have not been described for P. salmonis. Therefore, this study aimed to analyze the biofilm formation by P. salmonis isolates under several NaCl and iron concentrations and to evaluate the virulence of planktonic and sessile bacteria, together with the immune gene expression induced by these bacterial conditions in an Atlantic salmon macrophage cell line. Our results showed that NaCl and Fe significantly increased biofilm production in the LF-89 type strain and EM-90-like isolates. Additionally, the planktonic EM-90 isolate and sessile LF-89 generated the highest virulence levels, associated with differential expression of il-1β, il-8, nf-κb, and iκb-α genes in SHK-1 cells. These results suggest that there is no single virulence pattern or gene expression profile induced by the planktonic or sessile condition of P. salmonis, which are dependent on each strain and bacterial condition used. Full article

Piscirickettsia salmons, the causative agent of piscirickettsiosis, is genetically divided into two genomic groups, named after the reference strains as LF-89-like or EM-90-like. Phenotypic differences have been detected between the P. salmonis genogroups, including antibiotic susceptibilities, host specificities and pathogenicity. In this study, we aimed to develop a rapid, sensitive and cost-effective assay for the differentiation of the P. salmonis genogroups. Using an in silico analysis of the P. salmonis 16S rDNA digestion patterns, we have designed a genogroup-specific assay based on PCR-restriction fragment length polymorphism (RFLP). An experimental validation was carried out by comparing the restriction patterns of 13 P. salmonis strains and 57 field samples obtained from the tissues of dead or moribund fish. When the bacterial composition of a set of field samples, for which we detected mixtures of bacterial DNA, was analyzed by a high-throughput sequencing of the 16S rRNA gene amplicons, a diversity of taxa could be identified, including pathogenic and commensal bacteria. Despite the presence of mixtures of bacterial DNA, the characteristic digestion pattern of the P. salmonis genogroups could be detected in the field samples without the need of a microbiological culture and bacterial isolation. Full article

Piscirickettsia salmonis is the causative bacterial agent of piscirickettsiosis, a systemic fish disease that significantly impacts the Chilean salmon industry. This bacterium possesses a type IV secretion system (T4SS), several proteins of the type III secretion system (T3SS), and a single heat shock protein 60 (Hsp60/GroEL). It has been suggested that due to its high antigenicity, the P. salmonis Hsp60 could be surface-exposed, translocated across the membrane, and (or) secreted into the extracellular matrix. This study tests the hypothesis that P. salmonis Hsp60 could be located on the bacterial surface. Immunogold electron microscopy and proteomic analyses suggested that although P. salmonis Hsp60 was predominantly associated with the bacterial cell cytoplasm, Hsp60-positive spots also exist on the bacterial cell envelope. IgY antibodies against P. salmonis Hsp60 protected SHK-1 cells against infection. Several bioinformatics approaches were used to assess Hsp60 translocation by the T4SS, T3SS, and T6SS, with negative results. These data support the hypothesis that small amounts of Hsp60 must reach the bacterial cell surface in a manner probably not mediated by currently characterized secretion systems, and that they remain biologically active during P. salmonis infection, possibly mediating adherence and (or) invasion. Full article