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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-1ß, 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.

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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.

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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.

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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.

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Public health is facing a new challenge due to the increased bacterial resistance to most of the conventional antibacterial agents. Inadequate use of antibiotics in the Chilean aquaculture industry leads to the generation of multidrug resistance bacteria. Many fish pathogenic bacteria produce biofilm upon various sources of stress such as antibiotics, which provides several survival advantages for the bacterial life in community and can constitute a reservoir of pathogens in the marine environment. Being florfenicol a broad-spectrum antibiotic commonly used to treat infections in aquaculture, the aim of this study was to assess whether this antibiotic modulates in vitro the biofilm formation in several isolates of Piscirickettsia salmonis. Standard antibiotic-micro broth 96-flat well plates were used to determinate the minimal inhibitory concentration of florfenicol in eight different P. salmonis isolates. In vitro findings, with P. salmonis growing in the presence and absence of the antibiotic, exhibited a statistically significantly increase (p < .05) in biofilm formation in all the bacterial isolates cultivated with sub-MIC (defined as the half of the minimal inhibitory concentration in the presence of antibiotic) of florfenicol compared with controls (antibiotic-free broth). In conclusion, sub-MIC of florfenicol induced an increased biofilm formation in all P. salmonis isolates tested.

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Piscirickettsiosis is a fish disease caused by the Gram-negative bacterium Piscirickettsia salmonis. This disease has a high socio-economic impact on the Chilean salmonid aquaculture industry. The bacterium has a cryptic character in the environment and their main reservoirs are yet unknown. Bacterial biofilms represent a ubiquitous mechanism of cell persistence in diverse natural environments and a risk factor for the pathogenesis of several infectious diseases, but their microbiological significance for waterborne veterinary diseases, including piscirickettsiosis, have seldom been evaluated. This study analyzed the in vitro biofilm behavior of P. salmonis LF-89T (genogroup LF-89) and CA5 (genogroup EM-90) using a multi-method approach and elucidated the potential arsenal of virulence of the P. salmonis LF-89T type strain in its biofilm state. P. salmonis exhibited a quick kinetics of biofilm formation that followed a multi-step and highly strain-dependent process. There were no major differences in enzymatic profiles or significant differences in cytotoxicity (as tested on the Chinook salmon embryo cell line) between biofilm-derived bacteria and planktonic equivalents. The potential arsenal of virulence of P. salmonis LF-89T in biofilms, as determined by whole-transcriptome sequencing and differential gene expression analysis, consisted of genes involved in cell adhesion, polysaccharide biosynthesis, transcriptional regulation, and gene mobility, among others. Importantly, the global gene expression profiles of P. salmonis LF-89T were not enriched with virulence-related genes upregulated in biofilm development stages at 24 and 48 h. An enrichment in virulence-related genes exclusively expressed in biofilms was also undetected. These results indicate that early and mature biofilm development stages of P. salmonis LF-89T were transcriptionally no more virulent than their planktonic counterparts, which was supported by cytotoxic trials, which, in turn, revealed that both modes of growth induced important and very similar levels of cytotoxicity on the salmon cell line. Our results suggest that the aforementioned biofilm development stages do not represent hot spots of virulence compared with planktonic counterparts. This study provides the first transcriptomic catalogue to select specific genes that could be useful to prevent or control the (in vitro and/or in vivo) adherence and/or biofilm formation by P. salmonis and gain further insights into piscirickettsiosis pathogenesis.

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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.

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Bacterial cell envelopes play a critical role in host-pathogen interactions. Macromolecular components of these structures have been closely linked to the virulence of pathogens. Piscirickettsia salmonis is a relevant salmonid pathogen with a worldwide distribution. This bacterium is the etiological agent of piscirickettsiosis, a septicemic disease that causes a high economic burden, especially for the Chilean salmon farming industry. Although P. salmonis has been discovered long ago, its pathogenicity and virulence mechanisms are not completely understood. In this work, we present a genetic approach for producing in-frame deletion mutants on genes related to the biosynthesis of membrane-associated polysaccharides. We provide a detailed in vitro phenotype description of knock-out mutants on wzx and wcaJ genes, which encode predicted lipopolysaccharide (LPS) flippase and undecaprenyl-phosphate glucose phosphotransferase enzymes, respectively. We exhibit evidence that the wzx mutant strain carries a defect in the probably most external LPS moiety, while the wcaJ mutant proved to be highly susceptible to the bactericidal action of serum but retained the ability of biofilm production. Beyond that, we demonstrate that the deletion of wzx, but not wcaJ, impairs the virulence of P. salmonis in an intraperitoneally infected Atlantic salmon, Salmo salar, model of piscirickettsiosis. Our findings support a role for LPS in the virulence of P. salmonis during the onset of piscirickettsiosis.

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Piscirickettsiosis (SRS) has been the most important infectious disease in Chilean salmon farming since the 1980s. It was one of the first to be described, and to date, it continues to be the main infectious cause of mortality. How can we better understand the epidemiological situation of SRS? The catch-all answer is that the Chilean salmon farming industry must fight year after year against a multifactorial disease, and apparently only the environment in Chile seems to favor the presence and persistence of Piscirickettsia salmonis. This is a fastidious, facultative intracellular bacterium that replicates in the host's own immune cells and antigen-presenting cells and evades the adaptive cell-mediated immune response, which is why the existing vaccines are not effective in controlling it. Therefore, the Chilean salmon farming industry uses a lot of antibiotics-to control SRS-because otherwise, fish health and welfare would be significantly impaired, and a significantly higher volume of biomass would be lost per year. How can the ever-present risk of negative consequences of antibiotic use in salmon farming be balanced with the productive and economic viability of an animal production industry, as well as with the care of the aquatic environment and public health and with the sustainability of the industry? The answer that is easy, but no less true, is that we must know the enemy and how it interacts with its host. Much knowledge has been generated using this line of inquiry, however it remains insufficient. Considering the state-of-the-art summarized in this review, it can be stated that, from the point of view of fish immunology and vaccinology, we are quite far from reaching an effective and long-term solution for the control of SRS. For this reason, the aim of this critical review is to comprehensively discuss the current knowledge on the interaction between the bacteria and the host to promote the generation of more and better measures for the prevention and control of SRS.

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Piscirickettsiosis is the most important bacterial disease in the Chilean salmon industry, which has borne major economic losses due to failure to control it. Cells use extracellular vesicles (EVs) as an inter-cellular communicators to deliver several factors (e.g., microRNAs) that may regulate the responses of other cells. However, there is limited knowledge about the identification and characterization of EV-miRNAs in salmonids or the effect of infections on these. In this study, Illumina sequencing technology was used to identify Coho salmon plasma EV-miRNAs upon Piscirickettsia salmonis infection at four different time points. A total of 118 novels and 188 known EV-miRNAs, including key immune teleost miRNAs families (e.g., miR-146, miR-122), were identified. A total of 245 EV-miRNAs were detected as differentially expressed (FDR < 5%) in terms of control, with a clear down-regulation pattern throughout the disease. KEGG enrichment results of EV-miRNAs target genes showed that they were grouped mainly in cellular, stress, inflammation and immune responses. Therefore, it is hypothesized that P. salmonis could potentially benefit from unbalanced modulation response of Coho salmon EV-miRNAs in order to promote a hyper-inflammatory and compromised immune response through the suppression of different key immune host miRNAs during the course of the infection, as indicated by the results of this study.

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Piscirickettsia salmonis is the causative agent of Piscirickettsiosis, a systemic disease generating high mortality rates in farmed salmon cultures of southern Chile. Proteolytic enzymes are important virulence factors since they play a key role in bacterial invasion and proliferation within the host. Bacteria growing in muscle tissues are known to secrete proteases, but no proteolytic enzymes have been described in P. salmonis to date. A battery of putative protease genes was found in the genomes and available strains of P. salmonis by bioinformatics analyses, and their identity was established through comparison with protease genes in databases. The transcript levels of five candidate genes were analysed by in vitro infection and qPCR. All strains were found to generate protease activity to varying degrees, and this was significantly increased when bacteria infected a salmon cell line. Gene expression of several types of proteases was also evidenced, with the highest levels corresponding to the type 1 secretion system (T1SS), which is also involved in the transport of haemolysin A, although transcripts with significant levels of peptidase M4 (thermolysin) and CLP protease were also found.

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Piscirickettsiosis is the most important bacterial disease in the Chilean salmon industry, which has sorted several efforts to its control, generating enormous economic losses. Epigenetic alterations, such as DNA methylation, can play a relevant role in the modulation of the metazoans response to pathogens. Bacterial disease may activate global and local immune responses generating intricate responses with significant biological impact in the host. However, it is scarcely understood how bacterial infections influence fish epigenetic alterations. In the present study, we utilized Pacific salmon and Piscirickettsiosis as model, to gain understanding into the dynamics of DNA methylation among fish-bacterial infection interactions. A genome-wide analysis of DNA methylation patterns in female spleen tissue of Pacific salmon was achieved by reduced representation bisulphite sequencing from a time course design. We determined 2,251, 1,918, and 2,516 differentially methylated regions DMRs among infected and control Pacific salmon in 1 dpi, 5 dpi, and 15 dpi, respectively. The mean methylation difference per DMR among control and infected groups was of ~35%, with an oscillatory pattern of hypo, hyper, and hypomethylation across the disease. DMCs, among the control and infected group, showed that they were statistically enriched in intergenic regions and depleted in exons. Functional annotation of the DMR genes demonstrated three KEGG principal categories, associated directly with the host response to pathogens infections. Our results provide the first evidence of epigenetic variation in fish provoked by bacterial infection and demonstrate that this variation can be modulated across the disease.

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Piscirickettsiosis is a fish disease caused by the facultative intracellular bacterium, Piscirickettsia salmonis. Even though entry routes of P. salmonis in fish are not fully clear yet, the skin seems to be the main portal in some salmonid species. Despite the importance of fish mucous skin barrier in fighting waterborne pathogens, the interaction between salmonid skin mucus and the bacterium is unknown. This study seeks to determine the in vitro changes in the growth of two Chilean P. salmonis strains (LF-89-like and EM-90-like genotypes) and the type strain LF-89T under exposures to skin mucus from Salmo salar and Oncorhynchus mykiss, as well as changes in the cytotoxic effect of P. salmonis on the SHK-1 cells following exposures. The results suggest that the growth of three P. salmonis strains was not significantly negatively affected under exposures to skin mucus (adjusted at 100 µg total protein ml-1 ) of O. mykiss (69 ± 18 U lysozyme ml-1 ) and S. salar (48 ± 33 U lysozyme ml-1 ) over time. However, the cytotoxic effect of P. salmonis, pre-exposed to salmonid skin mucus, on the SHK-1 cell line was reliably identified only towards the end of the incubation period, suggesting that the mucus had a delaying effect on the cytotoxic response of the cell line to the bacterium. These results represent a baseline knowledge to open new avenues of research intended to understand how P. salmonis faces the fish mucous skin barrier.

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Piscirickettsia salmonis is a facultative intracellular bacterium that generates piscirickettsiosis affecting salmonids in Chile. The bacterium has the adaptability to survive in the marine environment under multiple stressful conditions. In this sense, this work focused on the analysis of a gene battery associated with biofilm formation under different culture conditions and on the adaptability of this biofilm to different media. The results indicated that the strains LF-89, IBM-034 and IBM-040 were strong biofilm producers, evidencing adaptability to the media by increasing the amount of biofilm through successive growths. Transcript levels of six genes described in various bacteria and P. salmonis, considered to have metabolic functions, and playing a relevant role in biofilm formation, were analyzed to evaluate bacterial functionality in the biofilm. The genes mazE-mazF, implicated in biofilm and stress, were markedly overexpressed in the biofilm condition in the three strains. For its part, gene gltA, an indicator of metabolic activity and related to virulence inhibition in Salmonella typhimurium, also seems to restrain the pathogenesis process in P. salmonis by inhibiting the expression of the virulence-associated genes liso and tcf. Finally, the expression of the glnA gene suggests the use of glutamine as an essential element for the growth of the biofilm.

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Early detection of piscirickettsiosis is an important purpose of government- and industry-based surveillance for the disease in Atlantic salmon farms in Chile. Real-time qPCRs are currently used for surveillance because bacterial isolation is inadequately sensitive or rapid enough for routine use. Since no perfect tests exist, we used Bayesian latent class models to estimate diagnostic sensitivity (DSe) and specificity (DSp) of qPCR and culture using separate two-test, single-population models for three farms (n = 148, 151, 44). Informative priors were used for DSp (culture (beta(999,1); qPCR (beta(98,2)), and flat priors (beta 1,1) for DSe and prevalence. Models were run for liver and kidney tissues combined and separately, based on the presence of selected gross-pathological signs. Across all models, qPCR DSe was 5- to 30-fold greater than for culture. Combined-tissue qPCR median DSe was highest in Farm 3 (sampled during P. salmonis outbreak (DSe = 97.6%)) versus Farm 1 (DSe = 85.6%) or Farm 2 (DSe = 83.5%), both sampled before clinical disease. Median DSe of qPCR was similar for liver and kidney, but higher when gross-pathological signs were evident at necropsy. High DSe and DSp and rapid turnaround-time indicate that the qPCR is fit for surveillance programmes and diagnosis during an outbreak. Targeted testing of salmon with gross-pathological signs can enhance DSe.

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The aetiological agent of Piscirickettsiosis is Piscirickettsia salmonis, a Gram-negative intracellular pathogen, and high doses of antibiotics have regularly been employed to treat this infection. Seven florfenicol and/or oxytetracycline resistance genes (tet pump, tetE, Tclor/flor, Tbcr, TfloR, ompF and mdtN) were identified in strains by in silico genome analyses. Later, the number of single nucleotide polymorphisms (SNPs) and its relationship with the resistance to these antibiotics were identified and analysed, using the original LF-89 strain as reference. Trials to determine and compare the minimum inhibitory concentration (MIC) of oxytetracycline and florfenicol in each strain, as well as to quantify the gPCR transcripts levels in the selected genes, were performed. Therefore, variations in the resistance to both antibiotics were observed, where the strain with fewer SNPs showed the highest susceptibility. Consistently, the in silico 3D analyses of proteins encoded by the selected genes revealed structural changes, evident in the sequences with the highest number of SNPs. These results showed that the bacterial resistance to oxytetracycline was mainly linked to the presence of SNPs in relevant sites, antibiotic resistance genes and an OmpF porin, leading to important changes in the protein structure.

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Piscirickettsia salmonis is the etiologic agent of salmon rickettsial syndrome (SRS) and is responsible for considerable economic losses in salmon aquaculture. The bacterium affects coho salmon (CS; Oncorhynchus kisutch), Atlantic salmon (AS; Salmo salar), and rainbow trout (RT; Oncorhynchus mykiss) in several countries, including Norway, Canada, Scotland, Ireland, and Chile. We used Bayesian genome-wide association study analyses to investigate the genetic architecture of resistance to P. salmonis in farmed populations of these species. Resistance to SRS was defined as the number of days to death and as binary survival (BS). A total of 828 CS, 2130 RT, and 2601 AS individuals were phenotyped and then genotyped using double-digest restriction site-associated DNA sequencing and 57K and 50K Affymetrix® Axiom® single nucleotide polymorphism (SNP) panels, respectively. Both traits of SRS resistance in CS and RT appeared to be under oligogenic control. In AS, there was evidence of polygenic control of SRS resistance. To identify candidate genes associated with resistance, we applied a comparative genomics approach in which we systematically explored the complete set of genes adjacent to SNPs, which explained more than 1% of the genetic variance of resistance in each salmonid species (533 genes in total). Thus, genes were classified based on the following criteria: i) shared function of their protein domains among species, ii) shared orthology among species, iii) proximity to the SNP explaining the highest proportion of the genetic variance, and iv) presence in more than one genomic region explaining more than 1% of the genetic variance within species. Our results allowed us to identify 120 candidate genes belonging to at least one of the four criteria described above. Of these, 21 of them were part of at least two of the criteria defined above and are suggested to be strong functional candidates influencing P. salmonis resistance. These genes are related to diverse biological processes, such as kinase activity, GTP hydrolysis, helicase activity, lipid metabolism, cytoskeletal dynamics, inflammation, and innate immune response, which seem essential in the host response against P. salmonis infection. These results provide fundamental knowledge on the potential functional genes underpinning resistance against P. salmonis in three salmonid species.

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Piscirickettsia salmonis is a facultative intracellular pathogen and etiological agent of the systemic disease salmonid rickettsial septicemia. It has been suggested that P. salmonis is able to survive in host macrophages, localized within a vacuole like-compartment which prevents lysosomal degradation. However, the relevant aspects of the pathogenesis of P. salmonis as the host modulation that allow its intracellular survival have been poorly characterized. In this study, we evaluated the role of lysosomes in the response to P. salmonis infection in macrophage-enriched cell cultures established from Atlantic salmon head kidneys. Bacterial infection was confirmed using confocal microscopy. A gentamicin protection assay was performed to recover intracellular bacteria and the 16S rDNA copy number was quantified through quantitative polymerase chain reaction in order to determine the replication of P. salmonis within macrophages. Lysosomal activity in Atlantic salmon macrophage-enriched cell cultures infected with P. salmonis was evaluated by analyzing the lysosomal pH and proteolytic ability through confocal microscopy. The results showed that P. salmonis can survive ≥120 h in Atlantic salmon macrophage-enriched cell cultures, accompanied by an increase in the detection of the 16S rDNA copy number/cell. The latter finding suggests that P. salmonis also replicates in Atlantic salmon macrophage-enriched cell cultures. Moreover, this bacterial survival and replication appears to be favored by a perturbation of the lysosomal degradation system. We observed a modulation in the total number of lysosomes and lysosomal acidification following infection with P. salmonis. Collectively, the results of this study showed that infection of Atlantic salmon macrophages with P. salmonis induced limited lysosomal response which may be associated with host immune evasion mechanisms of P. salmonis that have not been previously reported.

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