Flavobacterium psychraquaticum sp. nov., isolated from water system of Atlantic salmon (Salmo salar) smolts cultured in Chile.

Strain LB-N7T, a novel Gram-negative, orange, translucent, gliding, rod-shaped bacterium, was isolated from water samples collected from an open system of Atlantic salmon (Salmo salar) smolts in a fish farm in Chile during a flavobacterial infection outbreak in 2015. Phylogenetic analysis based on 16S rRNA sequences (1337 bp) revealed that strain LB-N7T belongs to the genus Flavobacterium and is closely related to the type strains Flavobacterium ardleyense A2-1T (98.8 %) and Flavobacterium cucumis R2A45-3T (96.75 %). The genome size of strain LB-N7T was 2.93 Mb with a DNA G+C content 32.6 mol%. Genome comparisons grouped strain LB-N7T with Flavobacterium cheniae NJ-26T, Flavobacterium odoriferum HXWNR29T, Flavobacterium lacisediminis TH16-21T and Flavobacterium celericrescens TWA-26T. The calculated digital DNA-DNA hybridization values between strain LB-N7T and the closest related Flavobacterium strains were 23.3 % and the average nucleotide identity values ranged from 71.52 to 79.39 %. Menaquinone MK-6 was the predominant respiratory quinone, followed by MK-7. The major fatty acids were iso-C15 : 0 and anteiso-C15 : 0. The primary polar lipids detected included nine unidentified lipids, two amounts of aminopospholipid and phospholipids, and a smaller amount of aminolipid. Phenotypic, genomic, and chemotaxonomic data suggest that strain LB-N7T (=CECT 30406T=RGM 3221T) represents as a novel bacterial species, for which the name Flavobacterium psychraquaticum sp. nov. is proposed.

Phenotypic and genomic characterization of a non-pathogenic Epilithonimonas ginsengisoli isolated from diseased farmed rainbow trout (Oncorhynchus mykiss) in Chile.

Flavobacterial infection associated with diseased fish is caused by multiple bacterial species within the family Flavobacteriaceae. In the present study, the Chilean isolate FP99, from the gills of a diseased, farmed rainbow trout (Oncorhynchus mykiss), was characterized using phenotypic and genomic analyses. Additionally assessed was pathogenic activity. Phylogenetic analysis based on 16S rRNA gene sequencing confirmed that isolate FP99 belonged to the genus Epilithonimonas, an average nucleotide identity value of 100% was detected with the Chilean isolate identified as Epilithonimonas sp. FP211-J200. In silico genome analysis, mechanisms for toxins production, and superantigens, adhesion, or other genes associated with virulence were not detected. However, genes encoding proteins for antibiotic resistance were found, including the chrA gene and the nucleotide sequence that encodes for multiple antibiotic resistance MarC proteins. Furthermore, the blaESP-1 gene (87.85% aminoacidic sequence identity), encoding an extended-spectrum subclass B3 metallo-ß-lactamase and conferring carbapenem-hydrolysing activity, and the tet(X) gene, which encodes a monooxygenase that catalyses the degradation of tetracycline-class antimicrobials were carried by this isolate. Phenotyping analyses also supported assignment as E. ginsengisoli. Challenge trials against healthy rainbow trout resulted in no observed pathogenic effect. Our findings identify for the first time the species E. ginsengisoli as associated with fish farming, suggesting that this isolate may be a component of the microbiota of the freshwater system. Notwithstanding, poor environmental conditions and any stressors associated with aquaculture situations or lesions caused by other pathogenic bacteria, such as F. psychrophilum, could favour the entry of E. ginsengisoli into rainbow trout.

Tenacibaculum bernardetii sp. nov., isolated from Atlantic salmon (Salmo salar L.) cultured in Chile.

Strain PVT-9aT, a novel Gram-stain-negative, aerobic, non-spore-forming, motile-by-gliding and rod-shaped bacterium, was isolated from a skin lesion of Atlantic salmon (Salmo salar L.) during a tenacibaculosis outbreak that occurred in 2016 at a Chilean fish farm. Phylogenetic analysis based on 16S rRNA gene sequencing confirmed that strain PVT-9aT belonged to the genus Tenacibaculum, being related to the closest type strains Tenacibaculum haliotis KCTC 52419T (98.49 % sequence similarity), Tenacibaculum aestuariivivum JDTF-79T (97.36 %), Tenacibaculum insulae JDTF-31T (97.29 %) and Tenacibaculum ovolyticum IFO 15947T (97.15 %). The genome size of strain PVT-9aT was 2.73 Mb with a DNA G+C content 31.09 mol%. Average nucleotide identity analysis among 30 Tenacibaculum species rendered the most similar strains as follows: T. haliotis KCTC 52419T (87.91 %), T. ovolyticum IFO 15947T (82.47 %), Tenacibaculum dicentrarchi 35/09T (81.08 %), Tenacibaculum finnmarkense gv finnmarkense TNO006T (80.91 %) and T. finnmarkense gv ulcerans TNO010T (80.96 %). Menaquinone MK-6 was the predominant respiratory quinone. The predominant cell fatty acids (>10 %) were iso-C15 : 0, iso-C15 : 1 G and iso-C15 : 0 3-OH. Phenotypic, chemotaxonomic and genomic data supported the assignment of strain PVT-9aT (=DSM 115155T=RGM 3472T) as representing a novel species of Tenacibaculum, for which the name Tenacibaculum bernardetii sp. nov. is proposed.

In vitro phenotypic evidence for the utilization of iron from different sources and siderophores production in the fish pathogen Tenacibaculum dicentrarchi.

Iron uptake during infection is an essential pathogenicity factor of several bacteria, including Tenacibaculum dicentrarchi, an emerging pathogen for salmonid and red conger eel (Genypterus chilensis) farms in Chile. Iron-related protein families were recently found in eight T. dicentrarchi genomes, but biological studies have not yet confirmed functions. The investigation reported herein clearly demonstrated for the first time that T. dicentrarchi possesses different systems for iron acquisition-one involving the synthesis of siderophores and another allowing for the utilization of heme groups. Using 38 isolates of T. dicentrarchi and the type strain CECT 7612T , all strains grew in the presence of the chelating agent 2.2'-dipyridyl (from 50 to 150 µM) and produced siderophores on chrome azurol S plates. Furthermore, 37 of the 38 T. dicentrarchi isolates used at least four of the five iron sources (i.e. ammonium iron citrate, ferrous sulfate, iron chloride hexahydrate, haemoglobin and/or hemin) when added to iron-deficient media, although the cell yield was less when using hemin. Twelve isolates grew in the presence of hemin, and 10 of them used only 100 µM. Under iron-supplemented or iron-restricted conditions, whole cells of three isolates and the type strain showed at least one membrane protein induced in iron-limiting conditions (c.a. 37.9 kDa), regardless of the isolation host. All phenotypic results were confirmed by in-silico genomic T. dicentrarchi analysis. Future studies will aim to establish a relationship between iron uptake ability and virulence in T. dicentrarchi through in vivo assays.

Co-occurrence of heterogeneous Flavobacterium psychrophilum isolates within the same Chilean farm and during the same infectious outbreak.

Flavobacterium psychrophilum is a pathogenic bacterium affecting Chilean salmonid farms. High antigenic and genetic diversity exists among Chilean F. psychrophilum isolates, but the distribution thereof among farms is poorly understood. These epidemiological data are key for developing isolate-specific vaccines. The present study isolated F. psychrophilum in diseased Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss) from five freshwater farms between 2018 and 2019. Each farm only raised one salmonid species and was geographically separated from and did not share culturing water with the other farms. Antigenic and genetic analyses were conducted to shed light on the possibility of isolates coexisting within the same farm during outbreaks. A total of 68 Chilean F. psychrophilum isolates were recovered from skin lesions, gills, fins, kidney and spleen of moribund and live fish. Among the 68 Chilean isolates, mPCR-serotyping indicated three major serotypes (i.e. 23.5% type 0; 47.1% type 2; and 26.5% type 4) and, to a lesser degree, serotype 1 (2.9%). Sixteen antigenic groups were detected by slide agglutination. Genetic characterizations by 16S rRNA alleles identified 71% of the isolates as the virulent genogroup CSF259-93 allele. A predominant serotype was associated with each farm, with types 0 and 4 related to Atlantic salmon and types 1 and 2 to rainbow trout. Notwithstanding, several antigenic groups coexisted within some farms. Likewise, the experimental intramuscular challenges (n = 20) demonstrated that the type-2 isolates from rainbow trout were the most pathogenic among isolates recovered from infectious outbreaks in Atlantic salmon, especially as compared to those from types-0 and -4. These results allow us to suggest that prevention measures, specifically vaccines, should be developed according to dominant isolates and with specificity to each farm, that is the use of autogenous or site-specific vaccines.

Flavobacterium pygoscelis sp. nov., isolated from a chinstrap penguin chick (Pygoscelis antarcticus).

Strain I-SCBP12nT, a novel Gram-stain-negative, aerobic, non-spore-forming, motile-by-gliding and rod-shaped bacterium, was isolated from a chinstrap penguin chick (Pygoscelis antarcticus) during a 2015 expedition to the Chilean Antarctic territory. Phylogenetic analysis based on 16S rRNA gene sequencing confirmed that strain I-SCBP12nT belonged to the genus Flavobacterium, being closely related to strains Flavobacterium chryseum P3160T (98.52 %), Flavobacterium hercynium WB 4.2-33T (98.47 %) and Flavobacterium chilense LM-19-FpT (98.47 %). The genome size of strain I-SCBP12nT was 3.69 Mb with DNA G+C content 31.95 mol%. Genomic comparisons of strain I-SCBP12nT with type species in the genus Flavobacterium were performed, with obtained average values near 75.17 and 84.33 % for the blast and MUMer analyses of average nucleotide identity, respectively, and 0.86 for the tetranucleotides frequency analysis. These values are far from the accepted species cut-off values. Strain I-SCBP12nT contained MK-6 as the predominant menaquinone and the major polar lipids were aminophospholipid, an unidentified aminolipid and unidentified lipids. The predominant fatty acids (> 5 %) were iso-C14 : 0, iso-C15 : 0, anteiso-C15 : 0, iso-C16 : 0, iso-C16 : 1, iso-C16 : 0 3-OH, C15 : 1 ω6c and summed feature 3 (C16 : 1 ω7c/C16 : 1 ω6c). Phenotypic, chemotaxonomic and genomic data supported the assignment of strain I-SCBP12nT (=CECT 30404T=RGM 3223T) to a novel species of Flavobacterium, for which the name Flavobacterium pygoscelis sp. nov.is proposed.

Healthy and infected Atlantic salmon (Salmo salar) skin-mucus response to Tenacibaculum dicentrarchi under in vitro conditions.

Tenacibaculosis caused by Tenacibaculum dicentrarchi is the second most important bacterial disease that affects the Chilean salmon industry. The impacted fish show severe external gross skin lesions on different areas of the body. The external mucus layer that covers fish skin contains numerous immune substances that act as one of the main defense barriers against microbial colonization and invasions by potential pathogens. The present in vitro study aimed to evaluate and elucidate the role of the external mucus layer in the susceptibility of Atlantic salmon (Salmo salar) to three Chilean T. dicentrarchi strains and the type strain. For this, mucus collected from healthy and diseased (i.e., with T. dicentrarchi) Atlantic salmon were used, and various antibacterial and inflammatory parameters were analysed. The T. dicentrarchi strains were attracted to the mucus of Atlantic salmon regardless of health status. All four strains adhered to the skin mucus and very quickly grew using the mucus nutrients. Once infection was established, different mucosal defense components were activated in the fish, but the levels of bactericidal activity and of other enzymes were insufficient to eliminate T. dicentrarchi. Alternatively, this pathogen may be able to neutralize or evade these mechanisms. Therefore, the survival of T. dicentrarchi in fish skin mucus could be relevant to facilitate the colonization and subsequent invasion of hosts. The given in vitro results suggest that greater attention should be given to fish skin mucus as a primary defense against T. dicentrarchi.

Development of a PCR assay for the specific identification of the fish pathogen Tenacibaculum piscium.

Tenacibaculosis is an emerging disease that severely affects salmonid farming in Chile, producing high mortalities and causing great economic losses. This work describes a novel PCR assay for the specific detection of Tenacibaculum piscium, a species recently described and identified in tenacibaculosis outbreaks in Norway and Chile. The designed primers amplified a 678-bp fragment of the peptidase gene (peptidase M23 family) from T. piscium. This method is specific for T. piscium; no other chromosomal DNA amplification products were obtained for other Tenacibaculum species. In pure cultures, the PCR assay detected up to 500 pg of DNA, or the equivalent of 2.44 ± 0.06 × 104 CFU/ml. For seeded fish samples (i.e., gills, liver, kidney, and mucus), the sensitivity limit was 4.88 ± 0.11 × 106 CFU/g, sufficient to detect T. piscium in acute infections in fish. Notably, this sensitivity level was 100-fold lower for DNA extracted from mucus samples. As compared to other existing methodologies (e.g., gene sequencing), the PCR approach described in this work allowed for the easiest detection of T. piscium in mucus samples obtained from challenged fish, an important outcome considering that the identification of this bacterium is difficult. Our results indicate that the designed specific primers and PCR method provide a rapid and specific diagnosis of T. piscium.

Draft Genome Sequence of Tenacibaculum haliotis Strain RA3-2T, Isolated from Korean Wild Abalone (Haliotis discus hannai).

Here, we present the draft genome sequence of Tenacibaculum haliotis strain RA3-2T (i.e., KCTC 52419T and NBRC 112382T), isolated from Korean wild abalone (Haliotis discus hannai). As the only strain for this Tenacibaculum species worldwide, the information is of use for comparative genomic analyses delineating Tenacibaculum species.

Co-existence of two Yersinia ruckeri biotypes and serotype O1a retrieved from rainbow trout (Oncorhynchus mykiss) farmed in Puno, Peru.

Yersinia ruckeri causes important economic losses for rainbow trout (Oncorhynchus mykiss) farms worldwide. This bacterial disease is likely the most common among trout in Peru; however, no commercial vaccine is available nationally, which is, in part, due to a lack of information on the bacterium. The aim of the current study was to characterize 29 Y. ruckeri isolates sampled from seven cage-reared farms in the Puno Region, the focal point for aquaculture activities in Peru. For this, samples were taken from fish with clinical signs (i.e. haemorrhages, uni- or bilateral exophthalmia, hyphaemia and/or melanosis). Notable among our findings was the existence of both Y. ruckeri biotype 1 (9 isolates) and biotype 2 (20 isolates; negative for sorbitol and Tween 80). The isolates further differed in API profiles 5307100 (21 isolates), 1307100 (4 isolates), 1305100 (2 isolates), 1307120 (1 isolate) and 5305100 (1 isolate), with the main differences being in the tests for lysine decarboxylase, gelatine hydrolysis and D-saccharose fermentation. Despite these differences, all isolates shared identical ERIC-PCR and REP-PCR profiles and belonged to the O1a serotype. Fingerprints were identical to the reference strain CECT 955 (serotype O1a). The information obtained will be used for epidemiological purposes by health authorities and for the development of a vaccine against Y. ruckeri, a prominent request made by fish farmers in Peru.

Collective behavior and virulence arsenal of the fish pathogen Piscirickettsia salmonis in the biofilm realm.

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.

Comparative polyphasic characterization of Weissella strains isolated from beaked whale and rainbow trout (Oncorhynchus mykiss): confirmation of Weissella ceti sp. nov. and description of the novel Weissella tructae sp. nov. isolated from farmed rainbow trout.

The weissellosis agent bacterium (WS08T = CBMAI 2730) was isolated from diseased rainbow trout (Oncorhynchus mykiss) in Brazil. The whole genome sequence of this strain was compared with the Mexican W-1 strain, also isolated from diseased rainbow trout, and with the Weissella ceti type strain CECT 7719 T (= 1119-1A-09 T = CCUG 59653 T), recovered from the beaked whale. Digital DNA-DNA hybridization pairwise analyses scored 98.7% between the Mexican W-1 and Brazilian WS08T but just 24.4% for both fish isolates compared to the W. ceti type strain CECT 7719 T. The 16S rRNA gene sequence comparisons with isolates of W. ceti, available at GenBank, were conducted. All rainbow trout-pathogenic isolates grouped close (97% bootstrap confirmation), but when this group was compared to the W. ceti type strain CECT 7719 T the similarity varied from 78.9 to 79.1%. Phenotypic assays were also conducted, and the W. ceti type strain diverged from WS08T and W-1 in the hydrolysis of aesculin, D-mannose, and potassium gluconate and in the hydrolysis of hippurate. Moreover, WS08T and W-1 showed weak growth at 5 °C whereas no growth was observed for W. ceti CECT 7719 T. The major fatty acids (> 10% total fatty acids) presented by WS08T and W-1 were summed feature 8 (C18:1 ω7c/C18:1 ω6c), summed feature 3 (C16:1 ω6c/C16:1ω7c), and C16:0. The results of phylogenetic and phenotypic analyses clearly differentiated the W. ceti CECT 7719 T type strain from the assessed pathogenic strains obtained from rainbow trout. Therefore, Weissella strains isolated from rainbow trout, here represented by strain WS08T (= CBMAI 2730), should be known as members of a novel species for which the name Weissella tructae sp. nov. is proposed.

Isolation, identification, virulence potential and genomic features of Tenacibaculum piscium isolates recovered from Chilean salmonids.

Tenacibaculum piscium, a gram-negative bacterium isolated from the skin ulcers of sea-farmed fish, has only been described in Norway. In the present study, we examined 16 Chilean Tenacibaculum isolates recovered from different organs in moribund and dead Atlantic salmon (Salmo salar), Rainbow trout (Oncorhynchus mykiss) and Coho salmon (Oncorhynchus kisutch) cultured at different fish farms between 2014 and 2018. The present study applied biochemical, phenotypic, fatty acid and whole-genome sequence-based analyses to confirm the taxonomic status of the Chilean isolates. The obtained results are the first to confirm the presence of T. piscium in Chile and in Coho salmon, thus extending the recognized geographical and species distribution of this bacterium. Subsequent bath-challenge assays in Atlantic salmon utilizing three T. piscium isolates obtained from different hosts resulted in low cumulative mortality (i.e. 0-35%), even after exposure to an unnaturally high concentration of bacterial cells (i.e. > 107 cells/ml). However, scale loss and frayed fins were observed in dead fish. In silico whole-genome analysis detected various genes associated with iron acquisition, encoding of the type IX secretion system and cargo proteins, resistance to tetracycline and fluoroquinolones and stress responses. These data represent an important milestone towards a better understanding on the genomic repertoire of T. piscium.

Draft Genome Sequence of Tenacibaculum ovolyticum To-7Br, Recovered from a Farmed Atlantic Salmon (Salmo salar).

We present the draft genome sequence of Tenacibaculum ovolyticum isolate To-7Br, recovered from a gill of a farmed specimen of Atlantic salmon (Salmo salar) showing signs of tenacibaculosis. This study provides the first detailed insights into the genomic characteristics of T. ovolyticum isolated for the first time from fish farmed in Chile.

Evaluation of the in vitro susceptibility of Tenacibaculum dicentrarchi to tiamulin using minimum inhibitory concentration tests.

Tenacibaculosis caused by Tenacibaculum dicentrarchi is the third most important bacterial fish infection affecting the Chilean salmon industry. Losses to this disease are most frequently controlled by treatments with florfenicol and oxytetracycline. However, recent tenacibaculosis outbreaks were controlled through the extra-label, oral administration of tiamulin, resulting in high treatment efficiency. In this study, we present an analysis of susceptibility patterns of 32 T. dicentrarchi isolates and the type strain CECT 7612T to tiamulin by determining the minimum inhibitory concentrations (MICs) according to the procedures recommended by the Clinical and Laboratory Standard Institute, but fixing incubation temperature to the more appropriate for the growth of T. dicentrarchi (18 ºC). The MICs of the T. dicentrarchi isolates were unimodally distributed (0.06-1.0 µg/ml range), while the CECT 7612T strain presented an MIC of 0.5 µg/ml. Calculations using Normalized Resistance Interpretation provided epidemiological cut-off values of ≤1.0 µg/ml, with the 33 T. dicentrarchi classified as wild type. In Chile, tiamulin is authorized for use in other livestock species, but application in salmonids is extra-label. Our presented in vitro results suggest that tiamulin is a viable alternative to florfenicol, specifically as tiamulin requires comparatively lower concentrations to inhibit T. dicentrarchi. Considering that tiamulin is also exclusively for veterinary use, is classified as "least important" by the World Health Organization and has not resulted in the development of bacterial resistance, pharmaceutical companies should be requested to register tiamulin and provide alternative antimicrobial treatments for the salmonid industry.

Comparison between genome sequences of Chilean Tenacibaculum dicentrarchi isolated from red conger eel (Genypterus chilensis) and Atlantic salmon (Salmo salar) focusing on bacterial virulence determinants.

Tenacibaculum dicentrarchi is an emerging pathogen for salmonid cultures and red conger eel (Genypterus chilensis) in Chile, causing high economic losses not only in Chile but also to the global salmon industry. Infected fish show severe gross skin lesions that are sometimes accompanied by bone exposure. Despite pathogenicity demonstrated by Koch's postulates, no knowledge is currently available regarding the virulence machinery of T. dicentrarchi strains. Comparisons between the genome sequences of the eight T. dicentrarchi strains obtained from G. chilensis and Atlantic salmon (Salmo salar) provide insights on the existence of genomic diversity within this bacterium. The T. dicentrarchi type strain 3509T was used as a reference genome. Depending on the T. dicentrarchi strain, the discovered diversity included genes associated with iron acquisition mechanisms, copper homeostasis encoding, resistance to tetracycline and fluoroquinolones, pathogenic genomic islands and phages. Interestingly, genes encoding the T9SS membrane protein PorP/SprF were retrieved in all of the analysed T. dicentrarchi strains, regardless of the host fish (i.e. red conger eel or Atlantic salmon). However, the T6SS core component protein VgrG was identified in only one Atlantic salmon strain. Three types of peptidase genes and proteins associated with quorum sensing were detected in all of the T. dicentrarchi strains. In turn, all eight strains presented a total of 17 proteins associated with biofilm formation, which was previously confirmed through physiological studies. This comparative analysis will help elucidate and describe the genes and pathways that are likely involved in the virulence process of T. dicentrarchi. All or part of these predicted genes could aid the pathogen during the infective process in fish, making further physiological research necessary for clarification.

First report and characterization of Tenacibaculum maritimum isolates recovered from rainbow trout (Oncorhynchus mykiss) farmed in Chile.

The present study reports on the first isolation of Tenacibaculum maritimum in rainbow trout (Oncorhynchus mykiss) farmed in Chile. In March 2020, two cages raising rainbow trout (~250 g) in the Los Lagos Region suffered a disease outbreak. In total, 17,554 fish died (3.5%-4.8% accumulated mortality). Microbiological analysis of the diseased fish obtained two representative isolates (i.e. Tm-035 and Tm-036). These were obtained from the external gross skin lesions-typical of tenacibaculosis-of two fish. Phenotyping, PCR tests and sequencing of the 16S rRNA and housekeeping genes confirmed the isolates as T. maritimum. The pathogenic potential of Tm-035 was further assessed by bath challenging Atlantic salmon (Salmo salar), which killed 70 ± 15% of fish within 11 days. Dead fish presented the same external clinical signs as did the farmed rainbow trout specimens. This research further broadens the known host distribution of this pathogen. Furthermore, the virulence experiments demonstrated that T. maritimum does not have a specific host. Additional studies are needed to evaluate the risk of T. maritimum for the O. mykiss farming industry.

Evidence for the existence of extracellular vesicles in Renibacterium salmoninarum and related cytotoxic effects on SHK-1 cells.

Extracellular vesicles (EVs) in bacteria have been implicated in invasive and, through enzymes, infective processes. One Gram-positive bacterium lacking any EV research, despite having commercial impacts on the aquaculture industry, is Renibacterium salmoninarum. We addressed this gap in knowledge by utilizing scanning electron microscopy to provide the first reported evidence for the production of EVs by R. salmoninarum strain H-2. Dispersive light scattering detected that the EVs were heterogeneous in size, and the protein compositions were similar to the bacterial membrane and contained the virulent protein factors p22 and p57. The EVs additionally had a concentrated negative charge compared with R. salmoninarum H-2, as determined by Z potential. Finally, these particles seemed to play a role in host invasion in vitro in the salmon head kidney cell line, as demonstrated by the occurrence of a cytotoxic effect within the first 48 hr post-infection. Higher EV concentrations (i.e. 52.6 µg/ml) were more toxic than R. salmoninarum H-2. This information serves as a foundation to develop and test possible uses for R. salmoninarum EVs in salmon aquaculture, inspiring future advances against bacterial kidney disease.