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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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Piscirickettsiosis is the main cause of mortality in salmonids of commercial importance in Chile, which is caused by Piscirickettsia salmonis, a Gram-negative, γ-proteobacteria that can produce biofilm as one of its virulence factors. The Chilean salmon industry uses large amounts of antibiotics to control piscirickettsiosis outbreaks, which has raised concern about its environmental impact and the potential to induce antibiotic resistance. Thus, the use of phytogenic feed additives (PFA) with antibacterial activity emerges as an interesting alternative to antimicrobials. Our study describes the antimicrobial action of an Andrographis paniculate-extracted PFA on P. salmonis planktonic growth and biofilm formation. We observed complete inhibition of planktonic and biofilm growth with 500 and 400 µg/mL of PFA for P. salmonis LF-89 and EM-90-like strains, respectively. Furthermore, 500 µg/mL of PFA was bactericidal for both evaluated bacterial strains. Sub-inhibitory doses of PFA increase the transcript levels of stress (groEL), biofilm (pslD), and efflux pump (acrB) genes for both P. salmonis strains in planktonic and sessile conditions. In conclusion, our results demonstrate the antibacterial effect of PFA against P. salmonis in vitro, highlighting the potential of PFA as an alternative to control Piscirickettsiosis.

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Toll-like receptor 5 (TLR5) responds to the monomeric form of flagellin and induces the MyD88-depending signaling pathway, activating proinflammatory transcription factors such as NF-κB and the consequent induction of cytokines. On the other hand, HMGB1 is a highly conserved non-histone chromosomal protein shown to interact with and activate TLR5. The present work aimed to design and characterize TLR5 agonist peptides derived from the acidic tail of Salmo salar HMGB1 based on the structural knowledge of the TLR5 surface using global molecular docking platforms. Peptide binding poses complexed on TLR5 ectodomain model from each algorithm were filtrated based on docking scoring functions and predicted theoretical binding affinity of the complex. Circular dichroism spectra were recorded for each peptide selected for synthesis. Only intrinsically disordered peptides (6W, 11W, and SsOri) were selected for experimental functional assay. The functional characterization of the peptides was performed by NF-κB activation assays, RT-qPCR gene expression assays, and Piscirickettsia salmonis challenge in SHK-1 cells. The 6W and 11W peptides increased the nuclear translation of p65 and phosphorylation. In addition, the peptides induced the expression of genes related to the TLR5 pathway activation, pro- and anti-inflammatory response, and differentiation and activation of T lymphocytes towards phenotypes such as TH1, TH17, and TH2. Finally, it was shown that the 11W peptide protects immune cells against infection with P. salmonis bacteria. Overall, the results indicate the usefulness of novel peptides as potential immunostimulants in salmonids.

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Piscirickettsiosis is the most prevalent bacterial disease affecting seawater salmon in Chilean salmon industry. Antibiotic therapy is the first alternative to counteract infections caused by Piscirickettsia salmonis. The presence of bacterial biofilms on materials commonly used in salmon farming may be critical for understanding the bacterial persistence in the environment. In the present study, the CDC Biofilm Reactor® was used to investigate the effect of sub- and over-MIC of florfenicol on both the pre-formed biofilm and the biofilm formation by P. salmonis under the antibiotic stimuli on Nylon and high-density polyethylene (HDPE) surfaces. This study demonstrated that FLO, at sub- and over-MIC doses, decreases biofilm-embedded live bacteria in the P. salmonis isolates evaluated. However, it was shown that in the P. salmonis Ps007 strain the presence of sub-MIC of FLO reduced its biofilm formation on HDPE surfaces; however, biofilm persists on Nylon surfaces. These results demonstrated that P. salmonis isolates behave differently against FLO and also, depending on the surface materials. Therefore, it remains a challenge to find an effective strategy to control the biofilm formation of P. salmonis, and certainly other marine pathogens that affect the sustainability of the Chilean salmon industry.

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Recently, we showed that Atlantic salmon vaccinated against Piscirickettsia salmonis lose their protection upon coinfection with Caligus rogercresseyi (sea lice). However, the causes of the overriding effect of C. rogercresseyi infection have not been elucidated, and the molecular basis of the cellular and humoral immune responses upon C. rogercresseyi infection has not been described for vaccinated salmon. Therefore, we studied changes in the transcription of immune genes in vaccinated Atlantic salmon that were experimentally challenged by co-infecting them with C. rogercresseyi and P. salmonis. In general, coinfection treatments showed immune gene expression similar to treatments with a single P. salmonis infection, showing a decreased cellular response. However, a high variance was found between individual fish in the case of crucial cellular immune genes, with a few fish reacting overwhelmingly highly compared to the majority. This supports our previous findings on vaccination response variation and reinforces the idea that vaccination failures in the field might be caused by an overwhelming amount of vaccinated fish that display a deficient immune response to the infection.

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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.

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Piscirickettsia salmonis is one of the main pathogens causing considerable economic losses in salmonid farming. The DNA gyrase of several pathogenic bacteria has been the target of choice for antibiotic design and discovery for years, due to its key function during DNA replication. In this study, we carried out a combined in silico and in vitro approach to antibiotic discovery targeting the GyrA subunit of Piscirickettsia salmonis. The in silico results of this work showed that flumequine (-6.6 kcal/mol), finafloxacin (-7.2 kcal/mol), rosoxacin (-6.6 kcal/mol), elvitegravir (-6.4 kcal/mol), sarafloxacin (-8.3 kcal/mol), orbifloxacin (-7.9 kcal/mol), and sparfloxacin (-7.2 kcal/mol) are docked with good affinities in the DNA binding domain of the Piscirickettsia salmonis GyrA subunit. In the in vitro inhibition assay, it was observed that most of these molecules inhibit the growth of Piscirickettsia salmonis, except for elvitegravir. We believe that this methodology could help to significantly reduce the time and cost of antibiotic discovery trials to combat Piscirickettsia salmonis within the salmonid farming industry.

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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.

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Autophagy is a fundamental cellular process implicated in the health of the cell, acting as a cytoplasmatic quality control machinery by self-eating unfunctional organelles and protein aggregates. In mammals, autophagy can participate in the clearance of intracellular pathogens from the cell, and the activity of the toll-like receptors mediates its activation. However, in fish, the modulation of autophagy by these receptors in the muscle is unknown. This study describes and characterizes autophagic modulation during the immune response of fish muscle cells after a challenge with intracellular pathogen Piscirickettsia salmonis. For this, primary cultures of muscle cells were challenged with P. salmonis, and the expressions of immune markers il-1ß, tnfα, il-8, hepcidin, tlr3, tlr9, mhc-I and mhc-II were analyzed through RT-qPCR. The expressions of several genes involved in autophagy (becn1, atg9, atg5, atg12, lc3, gabarap and atg4) were also evaluated with RT-qPCR to understand the autophagic modulation during an immune response. In addition, LC3-II protein content was measured via Western blot. The challenge of trout muscle cells with P. salmonis triggered a concomitant immune response to the activation of the autophagic process, suggesting a close relationship between these two processes.

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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.

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Maintaining the high overall health of farmed animals is a central tenant of their well-being and care. Intense animal crowding in aquaculture promotes animal morbidity especially in the absence of straightforward methods for monitoring their health. Here, we used bacterial 16S ribosomal RNA gene sequencing to measure bacterial population dynamics during P. salmonis infection. We observed a complex bacterial community consisting of a previously undescribed core pathobiome. Notably, we detected Aliivibrio wodanis and Tenacibaculum dicentrarchi on the skin ulcers of salmon infected with P. salmonis, while Vibrio spp. were enriched on infected gills. The prevalence of these co-occurring networks indicated that coinfection with other pathogens may enhance P. salmonis pathogenicity.

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The development of fish oral vaccines is of great interest to the aquaculture industry due to the possibility of rapid vaccination of a large number of animals at reduced cost. In a previous study, we evaluated the effect of alginate-encapsulated Piscirickettsia salmonis antigens (AEPSA) incorporated in feed, effectively enhancing the immune response in Atlantic salmon (Salmo salar). In this study, we seek to characterize AEPSA produced by ionic gelation using an aerodynamically assisted jetting (AAJ) system, to optimize microencapsulation efficiency (EE%), to assess microparticle stability against environmental (pH, salinity and temperature) and gastrointestinal conditions, and to evaluate microparticle incorporation in fish feed pellets through micro-CT-scanning. The AAJ system was effective in obtaining small microparticles (d < 20 µm) with a high EE% (97.92%). Environmental conditions (pH, salinity and temperature) generated instability in the microparticles, triggering protein release. 62.42% of the protein content was delivered at the intestinal level after in vitro digestion. Finally, micro-CT-scanning images confirmed microparticle incorporation in fish feed pellets. In conclusion, the AAJ system is effective at encapsulating P. salmonis antigens in alginate with a high EE% and a size small enough to be incorporated in fish feed and produce an oral vaccine.

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Piscirickettsia salmonis is the etiological agent of Piscirickettsiosis, a severe disease that affects Atlantic salmon (Salmo salar) farmed in Chile and many other areas (Norway, Scotland, Ireland, Canada and the USA). This study investigated the effects of low-dose P. salmonis infection (1 × 102 CFU/ml) on Atlantic salmon. In this study, we challenged fish with an isolated representative of the EM-90 genogroup via intraperitoneal injection for 42 days. Infected fish displayed decreased haematocrit and haemoglobin levels at day 13 post-infection, indicating erythropenia, haemolysis and haemodilution. Conversely, their white blood cell counts increased on days 13 and 21 post-infection. Additionally, their iron levels decreased from day 2 post-infection, indicating iron deficiency and an inability to retrieve stored iron before infection. Their magnesium levels also decreased at day 28 post-infection, possibly due to osmoregulatory problems. Also, we observed an increase in lactate dehydrogenase activity on days 5, 21, and 28 post-infection, suggesting early symptoms of hepatotoxicity. Later analyses determined a decrease in plasma glucose levels from day 2 post-infection. This may be attributed to the hypoxic conditions caused by P. salmonis, leading to an excess utilization of stored carbohydrates. Our results suggest that the blood parameters we studied are useful for monitoring the physiological status of Atlantic salmon infected with P. salmonis.

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The study of the stress responses in bacteria has given us a wealth of information regarding the mechanisms employed by these bacteria in aggressive or even non-optimal living conditions. This information has been applied by several researchers to identify molecular targets related to pathogeny, virulence, and survival, among others, and to design new prophylactic or therapeutic strategies against them. In this study, our knowledge of these mechanisms has been summarized with emphasis on some aquatic pathogenic bacteria of relevance to the health and productive aspects of Chilean salmon farming (Piscirickettsia salmonis, Tenacibaculum spp., Renibacterium salmoninarum, and Yersinia ruckeri). This study will aid further investigations aimed at shedding more light on possible lines of action for these pathogens in the coming years.

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The innate immune system can limit the growth of invading pathogens by depleting micronutrients at a cellular and tissue level. However, it is not known whether nutrient depletion mechanisms discriminate between living pathogens (which require nutrients) and pathogen-associated molecular patterns (PAMPs) (which do not). We stimulated SHK-1 cells with different PAMPs (outer membrane vesicles of Piscirickettsia salmonis "OMVs", protein extract of P. salmonis "TP" and lipopolysaccharides of P. salmonis "LPS") isolated from P. salmonis and evaluated transcriptional changes in nutritional immunity associated genes. Our experimental treatments were: Control (SHK-1 stimulated with bacterial culture medium), OMVs (SHK-1 stimulated with 1µg of outer membrane vesicles), TP (SHK-1 stimulated with 1µg of total protein extract) and LPS (SHK-1 stimulated with 1µg of lipopolysaccharides). Cells were sampled at 15-, 30-, 60- and 120-minutes post-stimulation. We detected increased transcription of zip8, zip14, irp1, irp2 and tfr1 in all three experimental conditions and increased transcription of dmt1 in cells stimulated with OMVs and TP, but not LPS. Additionally, we observed generally increased transcription of ireg-1, il-6, hamp, irp1, ft-h and ft-m in all three experimental conditions, but we also detected decreased transcription of these markers in cells stimulated with TP and LPS at specific time points. Our results demonstrate that SHK-1 cells stimulated with P. salmonis PAMPs increase transcription of markers involved in the transport, uptake, storage and regulation of micronutrients such as iron, manganese and zinc.

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Research into Piscirickettsia salmonis biofilms on materials commonly used in salmon farming is crucial for understanding its persistence and virulence. We used the CDC Biofilm Reactor to investigate P. salmonis (LF-89 and EM-90) biofilm formation on Nylon, Stainless steel (316L), Polycarbonate and High-Density Polyethylene (HDPE) surfaces. After 144 h of biofilm visualization by scanning confocal laser microscopy under batch growth conditions, Nylon coupons generated the greatest biofilm formation and coverage compared to Stainless steel (316L), Polycarbonate and HDPE. Additionally, P. salmonis biofilm formation on Nylon was significantly greater (p ≤ .01) than Stainless steel (316L), Polycarbonate and HDPE at 288 h. We used Nylon coupons to determine the kinetic parameters of the planktonic and biofilm phases of P. salmonis. The two strains had similar latencies in the planktonic phase; however, LF-89 maximum growth was 2.5 orders of magnitude higher (Log cell ml-1 ). Additionally, LF-89 had a specified growth rate (µmax) of 0.0177 ± 0.006 h-1 and a generation time of 39.2 h. This study contributes to a deeper understanding of the biofilm formation by P. salmonis and elucidates the impact of the biofilm on aquaculture systems.

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The intensive salmon farming is associated with massive outbreaks of infections. The use of antibiotics for their prevention and control is related to damage to the environment and human health. Antimicrobial peptides (AMPs) have been proposed as an alternative to the use of antibiotics for their antimicrobial and immunomodulatory activities. However, one of the main challenges for its massive clinical application is the high production cost and the complexity of chemical synthesis. Thus, recombinant DNA technology offers a more sustainable, scalable, and profitable option. In the present study, using an AMPs function prediction methodology, we designed a chimeric peptide consisting of sequences derived from cathelicidin fused with the immunomodulatory peptide derived from flagellin. The designed peptide, CATH-FLA was produced by recombinant expression using an easy pre-purification system. The chimeric peptide was able to induce IL-1ß and IL-8 expression in Salmo salar head kidney leukocytes, and prevented Piscirickettsia salmonis-induced cytotoxicity in SHK-1 cells. These results suggest that pre-purification of a recombinant AMP-based chimeric peptide designed in silico allow obtaining a peptide with immunomodulatory activity in vitro. This could solve the main obstacle of AMPs for massive clinical applications.

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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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