Design and functional characterization of Salmo salar TLR5 agonist peptides derived from high mobility group B1 acidic tail.

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.

Effect of florfenicol on Piscirickettsia salmonis biofilm formed in materials used in salmonid nets, nylon and high-density polyethylene.

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.

In vitro effects of phytogenic feed additive on Piscirickettsia salmonis growth and biofilm formation.

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.

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

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

Antibiotic discovery against Piscirickettsia salmonis using a combined in silico and in vitro approach.

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.

Outer membrane vesicles from Piscirickettsia salmonis induce the expression of inflammatory genes and production of IgM in Atlantic salmon Salmo salar.

Piscirickettsiosis outbreaks due to Piscirickettsia salmonis occur globally in the Chilean salmon aquaculture generating significant monetary losses in the industry. P. salmonis secretes outer membrane vesicles (OMVs) which are naturally non-replicating and highly immunogenic spherical nanoparticles. P. salmonis OMVs has been shown to induce immune response in zebrafish; however, the immune response induced by these vesicles in salmonids has not been evaluated. In this study, we inoculated Atlantic salmon with 10 and 30 µg doses of P. salmonis OMVs and took samples for 12 days. qPCR analysis indicated an inflammatory response. Thus, the inflammatory genes evaluated were up- or down-regulated at several times in liver, head kidney and spleen. In addition, the liver was the organ most immune-induced, mainly in the 30 µg-dose. Interestingly, co-expression of pro- and anti-inflammatory cytokines was evidenced by the prominent expression of il-10 at day 1 in spleen and also in head kidney on days 3, 6 and 12, while il-10 and tgf-ß were up-regulated on days 3, 6 and 12 in liver. Importantly, we detected the production of IgM against proteins of P. salmonis in the serum collected from immunized fish after 14 days. Thus, 40 and 400 µg OMVs induced the production of highest IgM levels; however, no statistical difference in the immunoglobulin levels produced by these OMVs doses were detected. The current study provides evidence that OMVs released by P. salmonis induced a pro-inflammatory responses and IgM production in S. salar, while regulatory genes were induced in order to regulate their effects and achieve the balance of the inflammatory response.

Host-pathogen interaction involving cytoskeleton changes as well as non-coding regulation as primary mechanisms for SRS resistance in Atlantic salmon.

The salmonid rickettsial syndrome (SRS) is a systemic bacterial infection caused by Piscirickettsia salmonis that generates significant economic losses in Atlantic salmon (Salmo salar) aquaculture. Despite this disease's relevance, the mechanisms involved in resistance against P. salmonis infection are not entirely understood. Thus, we aimed at studying the pathways explaining SRS resistance using different approaches. First, we determined the heritability using pedigree data from a challenge test. Secondly, a genome-wide association analysis was performed following a complete transcriptomic profile of fish from genetically susceptible and resistant families within the challenge infection with P. salmonis. We found differentially expressed transcripts related to immune response, pathogen recognition, and several new pathways related to extracellular matrix remodelling and intracellular invasion. The resistant background showed a constrained inflammatory response, mediated by the Arp2/3 complex actin cytoskeleton remodelling polymerization pathway, probably leading to bacterial clearance. A series of biomarkers of SRS resistance, such as the beta-enolase (ENO-ß), Tubulin G1 (TUBG1), Plasmin (PLG) and ARP2/3 Complex Subunit 4 (ARPC4) genes showed consistent overexpression in resistant individuals, showing promise as biomarkers for SRS resistance. All these results together with the differential expression of several long non-coding RNAs show the complexity of the host-pathogen interaction of S. salar and P. salmonis. These results provide valuable information on new models describing host-pathogen interaction and its role in SRS resistance.

Immune response induced by coinfection of the sea louse Caligus rogercresseyi and the intracellular bacteria Piscirickettsia salmonis in vaccinated Atlantic salmon.

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.

The impact of Piscirickettsia salmonis infection on genome-wide DNA methylation profile in Atlantic Salmon.

Salmon rickettsial septicaemia (SRS), caused by the bacteria Piscirickettsia salmonis (P. salmonis), is responsible for significant mortality in farmed Atlantic salmon in Chile. Currently there are no effective treatments or preventive measures for this disease, although genetic selection or genome engineering to increase salmon resistance to SRS are promising strategies. The accuracy and efficiency of these strategies are usually influenced by the available biological background knowledge of the disease. The aim of this study was to investigate DNA methylation changes in response to P. salmonis infection in the head kidney and liver tissue of Atlantic salmon, and the interaction between gene expression and DNA methylation in the same tissues. The head kidney and liver methylomes of 66 juvenile salmon were profiled using reduced representation bisulphite sequencing (RRBS), and compared between P. salmonis infected animals (3 and 9 days post infection) and uninfected controls, and between SRS resistant and susceptible fish. Methylation was correlated with matching RNA-Seq data from the same animals, revealing that methylation in the first exon leads to an important repression of gene expression. Head kidney methylation showed a clear response to the infection, associated with immunological processes such as actin cytoskeleton regulation, phagocytosis, endocytosis and pathogen associated pattern receptor signaling. Our results contribute to the growing understanding of the role of methylation in regulation of gene expression and response to infectious diseases and could inform the incorporation of epigenetic markers into genomic selection for disease resistant and the design of diagnostic epigenetic markers to better manage fish health in salmon aquaculture.

Salmonid MyD88 is a key adapter protein that activates innate effector mechanisms through the TLR5M/TLR5S signaling pathway and protects against Piscirickettsia salmonis infection.

The membrane-anchored and soluble Toll-like Receptor 5 -TLR5M and TLR5S, respectively-from teleost recognize bacterial flagellin and induce the pro-inflammatory cytokines expression in a MyD88-dependent manner such as the TLR5 mammalian orthologous receptor. However, it has not been demonstrated whether the induced signaling pathway by these receptors activate innate effector mechanisms MyD88-dependent in salmonids. Therefore, in this work we study the MyD88 dependence on the induction of TLR5M/TLR5S signaling pathway mediated by flagellin as ligand on the activation of some innate effector mechanisms. The intracellular and extracellular Reactive Oxygen Species (ROS) production and conditioned supernatants production were evaluated in RTS11 cells, while the challenge with Piscirickettsia salmonis was evaluated in SHK-1 cells. Our results demonstrate that flagellin directly stimulates ROS production and indirectly stimulates it through the production of conditioned supernatants, both in a MyD88-dependent manner. Additionally, flagellin stimulation prevents the cytotoxicity induced by infection with P. salmonis in a MyD88-dependent manner. In conclusion we demonstrate that MyD88 is an essential adapter protein in the activation of the TLR5M/TLR5S signaling pathway mediated by flagellin in salmonids, which leads downstream to the induction of innate effector mechanisms, promoting immuno-protection against a bacterial challenge with P. salmonis.

New strategy for the design, production and pre-purification of chimeric peptide with immunomodulatory activity in Salmosalar.

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.

Stress response and virulence factors in bacterial pathogens relevant for Chilean aquaculture: current status and outlook of our knowledge.

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.

Piscirickettsia salmonis forms a biofilm on nylon surface using a CDC Biofilm Reactor.

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.

Effect of low-dose Piscirickettsia salmonis infection on haematological-biochemical blood parameters in Atlantic salmon (Salmo salar).

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.

Increased dietary availability of selenium in rainbow trout (Oncorhynchus mykiss) improves its plasma antioxidant capacity and resistance to infection with Piscirickettsia salmonis.

Salmonid Rickettsial Septicaemia (SRS), caused by Piscirickettsia salmonis, is the most important infectious disease in the Chilean salmon farming industry. An opportunity to control this disease is to use functional micronutrients to modulate host mechanisms of response to the infection. Since P. salmonis may affect the host antioxidant system in salmonids, particularly that dependent on selenium (Se), we hypothesized that fish's dietary selenium supplementation could improve the response to the bacterial infection. To address this, we defined a non-antibiotic, non-cytotoxic concentration of selenium to evaluate its effect on the response to in vitro infections of SHK-1 cells with P. salmonis. The results indicated that selenium supplementation reduced the cytopathic effect, intracellular bacterial load, and cellular mortality of SHK-1 by increasing the abundance and activity of host glutathione peroxidase. We then prepared diets supplemented with selenium up to 1, 5, and 10 mg/kg to feed juvenile trout for 8 weeks. At the end of this feeding period, we obtained their blood plasma and evaluated its ability to protect SHK-1 cells from infection with P. salmonis in ex vivo assays. These results recapitulated the observed ability of selenium to protect against infection with P. salmonis by increasing the concentration of selenium and the antioxidant capacity in fish's plasma. To the best of our knowledge, this is the first report of the protective capacity of selenium against P. salmonis infection in salmonids, becoming a potential effective host-directed dietary therapy for SRS and other infectious diseases in animals at a non-antibiotic concentration.

"Limiting access to iron decreases infection of Atlantic salmon SHK-1 cells with bacterium Piscirickettsia salmonis".

BACKGROUND: Vertebrate hosts limit the availability of iron to microbial pathogens in order to nutritionally starve the invaders. The impact of iron deficiency induced by the iron chelator deferoxamine mesylate (DFO) was investigated in Atlantic salmon SHK-1 cells infected with the facultative intracellular bacterium Piscirickettsia salmonis. RESULTS: Effects of the DFO treatment and P. salmonis on SHK-1 cells were gaged by assessing cytopathic effects, bacterial load and activity, and gene expression profiles of eight immune biomarkers at 4- and 7-days post infection (dpi) in the control group, groups receiving single treatments (DFO or P. salmonis) and their combination. The chelator appears to be well-tolerated by host cells, while it had a negative impact on the number of bacterial cells and associated cytotoxicity. DFO alone had minor effects on gene expression of SHK-1 cells, including an early activation of IL-1ß at 4 dpi. In contrast to few moderate changes induced by single treatments (either infection or chelator), most genes had highest upregulation in the infected groups receiving DFO. The mildest induction of hepcidin-1 (antimicrobial peptide precursor and regulator of iron homeostasis) was observed in cells exposed to DFO alone, followed by P. salmonis infected cells while the addition of DFO to infected cells further increased the mRNA abundance of this gene. Transcripts encoding TNF-α (immune signaling) and iNOS (immune effector) showed sustained increase at both time points in this group while cathelicidin-1 (immune effector) and IL-8 (immune signaling) were upregulated at 7 dpi. The stimulation of protective gene responses seen in infected cultures supplemented with DFO coincided with the reduction of bacterial load and activity (judged by the expression of P. salmonis 16S rRNA), and damage to cultured host cells. CONCLUSION: The absence of immune gene activation under normal iron conditions suggests modulation of host responses by P. salmonis. The negative effect of iron deficiency on bacteria likely allowed host cells to respond in a more protective manner to the infection, further decreasing its progression. Presented findings encourage in vivo exploration of iron chelators as a promising strategy against piscirickettsiosis.

Horizontal transmission of Piscirickettsia salmonis from the wild sub-Antarctic notothenioid fish Eleginops maclovinus to rainbow trout (Oncorhynchus mykiss) under experimental conditions.

Piscirickettsia salmonis is the aetiological agent of piscirickettsiosis, a bacterial disease that affects farmed salmonids, causing high mortalities and significant economic losses in the Chilean salmon farm industry. Given the Chilean native fish species Patagonian blenny, Eleginops maclovinus, lives in the vicinity of salmon farms, it is relevant to clarify the epidemiological role that this species could play in the transmission and/or dissemination of this pathogen. This study aimed to evaluate the bidirectional transmission of P. salmonis between the Patagonian blenny and Oncorhynchus mykiss (rainbow trout), via a cohabitation challenge model. The results of this study demonstrated the transmission of the bacteria from Patagonian blennies to rainbow trout, considering the specific mortality in cohabitant rainbow trout, reaching 46%: the necropsy of these specimens, evidencing the characteristic pathological lesions of the disease and the positive results of the qPCR analysis for P. salmonis, in the same individuals. In contrast, no mortalities of Patagonian blenny specimens were recorded in the challenged experimental groups. This study is the first report showing the horizontal transmission of P. salmonis from a native non-salmonid species, such as the Patagonian blenny, to a salmonid species, generating the disease and specific mortality in rainbow trout, using a cohabitation challenge.

Development of a Piscirickettsia salmonis immersion challenge model to investigate the comparative susceptibility of three salmon species.

Piscirickettsia salmonis, the aetiological agent of salmonid rickettsial septicaemia (SRS), is a global pathogen of wild and cultured marine salmonids. Here, we describe the development and application of a reproducible, standardized immersion challenge model to induce clinical SRS in juvenile pink (Oncorhynchus gorbuscha), Atlantic (Salmo salar) and sockeye salmon (O. nerka). Following a 1-hr immersion in 105 colony-forming units/ml, cumulative mortality in Atlantic salmon was 63.2% while mortality in sockeye salmon was 10%. Prevalence and levels of the bacterium in kidney prior to onset of mortality were lower in sockeye compared with Atlantic or pink salmon. The timing and magnitude of bacterial shedding were estimated from water samples collected during the exposure trials. Shedding was estimated to be 82-fold higher in Atlantic salmon as compared to sockeye salmon and peaked in the Atlantic salmon trial at 36 d post-immersion. These data suggest sockeye salmon are less susceptible to P. salmonis than Atlantic or pink salmon. Finally, skin lesions were observed on infected fish during all trials, often in the absence of detectable infection in kidney. As a result, we hypothesize that skin is the primary point of entry for P. salmonis during the immersion challenge.

Assessing the impacts of skin mucus from Salmo salar and Oncorhynchus mykiss on the growth and in vitro infectivity of the fish pathogen Piscirickettsia salmonis.

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.

First description and diagnostics of disease caused by Piscirickettsia salmonis in farmed European sea bass (Dicentrarchus labrax Linnaeus) from Croatia.

During the winter of 2013 and 2016, several Croatian fish farms experienced mortalities in the fry of European sea bass, Dicentrarchus labrax. Affected fish showed abnormal swimming behaviour and reduced appetite, and death ensued several days after the onset of clinical signs of disease. Necropsy revealed pale liver, empty digestive tract, distended gall bladder, and hyperaemia and congestion of the meninges. Routine bacteriological examination tested negative, and virological examination ruled out nodavirus infection. Histological examination revealed multifocal necrosis and extensive inflammation in the brain with abundant cellular debris in the ventricles. Inflammatory cells displayed intra-cytoplasmic basophilic vacuoles leading to suspicion of Piscirickettsia salmonis infection. Fluorescent in situ hybridization using an oligonucleotide probe targeting Domain Bacterium applied to tissue sections tested positive. The pathogen was identified by 16S rRNA gene sequencing of brain material, and the sequence showed 99% similarity with P. salmonis. This result enabled the design of an oligonucleotide probe specifically targeting P. salmonis. In 2016, P. salmonis was successfully isolated on CHAB from the brain of an affected specimen and identified using 16S rRNA gene sequencing and MALDI-TOF. This study describes the first outbreak of disease caused by P. salmonis in sea bass in Croatia, while new diagnostic tools will enable further research on its epidemiology and pathogenicity.