Outer membrane vesicles (OMVs) from Tenacibaculum maritimum as a potential vaccine against fish tenacibaculosis.

Outer membrane vesicles (OMVs) have been gained increasing attention in vaccinology due to their ability to induce strong protective humoral and cell-mediated immunity. The Gram-negative bacterium Tenacibaculum maritimum, the causative agent of marine tenacibaculosis, poses a significant challenge to the global aquaculture industry due to its difficult prophylaxis. In previous studies, we demonstrated that OMV production is a key virulence mechanism in T. maritimum. Building on this, the present study aimed to evaluate the efficacy of a natural, encapsulated multi-antigen vaccine made from adjuvant-free, crude T. maritimum OMVs (Tm-OMVs). A vaccination experiment using SP9.1-OMVs was conducted in juvenile turbot (Scophthalmus maximus L.), followed by a T. maritimum bath challenge. Immune responses in the turbot were assessed by measuring anti-Tm antibody levels and analyzing the expression of eight key immune-related genes (il-1ß, il-8, il-22, pcna, c3, cd4-1, ifng2, cd8α). The results showed that immunization with SP9.1-OMVs provided significant protection against T. maritimum infection (RPS = 70 %). Vaccinated fish exhibited a dose-dependent increase in anti-Tm antibody titers in blood plasma, along with rapid induction of both innate (il-1ß, il-8, il-22, c3) and adaptive (cd4-1, ifng2, cd8α) immune genes as early as 4 h post-bath challenge. These findings offer new insights into the early immune response of turbot following T. maritimum infection and could serve as a foundation for developing novel OMV-based vaccines.

Tenacibaculum dicentrarchi produce outer membrane vesicles (OMV) that are associated with the cytotoxic effect in rainbow trout head kidney macrophages.

Tenacibaculum dicentrarchi is the second most important pathogen in Chilean salmon farming. This microorganism causes severe skin lesions on the body surface of farmed fish. The bacterium can also adhere to surfaces and form biofilm, survive in fish skin mucus, and possess different systems for iron acquisition. However, the virulence mechanisms are still not fully elucidated. Outer membrane vesicles (OMV) are nanostructures released by pathogenic Gram-negative bacteria during growth, but none has been described yet for T. dicentrarchi. In this study, we provide the first reported evidence of the fish pathogen T. dicentrarchi producing and releasing OMV from 24 h after incubation, increasing thereafter until 120 h. Analyses were conducted with T. dicentrarchi TdCh05, QCR29, and the type strain CECT 7612T . The OMV sizes, determined via scanning electron microscopy, ranged from 82.25 nm to 396.88 nm as per the strain and incubation time point (i.e., 24 to 120 h). SDS-PAGE revealed that the number of protein bands evidenced a drastically downward trend among the T. dicentrarchi strains. In turn, the OMV shared five proteins (i.e., 22.2, 31.9, 47.7, 56.3, and 107.1 kDa), but no protein pattern was identical. A heterogeneous amount of protein, RNA, and DNA were obtained, depending on the time at which OMV were extracted. Purified OMV were biologically active and induced a cytotoxic effect in macrophage-enriched cell cultures from rainbow trout (Oncorhynchus mykiss) head kidneys. This is the first step towards understanding the role that OMV could play in the pathogenesis of T. dicentrarchi.

New Zealand rickettsia-like organism and Tenacibaculum maritimum vaccine efficacy study.

A cohort of Chinook salmon juveniles was vaccinated, with an autogenous bivalent vaccine against New Zealand RLOs (NZ-RLO1) and Tenacibaculum maritimum. A proportion of the cohort was not vaccinated to act as controls. At smoltification, the fish were challenged with NZ-RLO1, NZ-RLO2, or T. maritimum. We found that challenge with T. maritimum by immersion in (7.5 × 105 cfu/mL of water) did not yield any pathology. Challenge with RLOs produced clinical signs that were more or less severe depending on the challenge route, dose or vaccination status. Survival was significantly higher for vaccinated fish within the groups challenged with NZ-RLO1 by intraperitoneal injection with a relative percent survival (RPS) of 48.84%. Survival was not significantly different between vaccinated and non-vaccinated fish for groups challenged with NZ-RLO2 by intraperitoneal injection or by NZ-RLO1 by immersion. Yet, anecdotally the clinical disease presentation (manifesting as haemorrhagic, ulcerative skin lesions) was more severe for the non-vaccinated fish. This study demonstrates that autogenous vaccine against NZ-RLO is protective against severe disease and death by NZ-RLO1 challenge which warrants implementation and further evaluation under field conditions. Yet, this study also highlights the importance of the route of administration and dose when evaluating pathogenicity and vaccine efficacy.

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.

Tenacibaculum tangerinum sp. nov., isolated from a tidal flat sediment.

An orange-coloured bacterium, designated as strain GRR-S3-23T, was isolated from a tidal flat sediment collected from Garorim Bay, Chuncheongbuk-do, Republic of Korea. Cells of GRR-S3-23T were aerobic, Gram-stain-negative, rod-shaped and motile. GRR-S3-23T grew at 18-40 °C (optimum, 30 °C), pH 7.0-9.0 (optimum, pH 7.0) and with 2-4 % NaCl (optimum, 2-3 % w/v). Results of 16S rRNA gene sequence analysis indicated that GRR-S3-23T was closely related to Tenacibaculum aiptasiae a4T (97.6 %), followed by Tenacibaculum aestuarii SMK-4T (97.5 %), Tenacibaculum mesophilum MBIC 1140T (97.4 %), Tenacibaculum singaporense TLL-A2T (97.3 %), Tenacibaculum crassostreae JO-1T (97.2 %),and Tenacibaculum sediminilitoris YKTF-3T (97.1 %). The average amino acid identity values between GRR-S3-23T and the related strains were 86.8-72.8 %, the average nucleotide identity values were 83.3-74.1 %, and the digital DNA-DNA hybridization values were 27.0-19.6 %. GRR-S3-23T possessed menaquinone-6 (MK-6) as major respiratory quinone and had summed feature 3 (C16 : 1ω7c and/or C16 : 1ω6c, 20.6 %) and iso-C15 : 1G (10.8 %) as major fatty acids (>10.0 %). The polar lipid profiles of GRR-S3-23T contained phosphatidylethanolamine, one unidentified aminolipid, one unidentified aminophospholipid, three unidentified lipids, one unidentified glycolipid and four unidentified phospholipids. The DNA G+C content of GRR-S3-23T was 33.7%. On the basis of the results of the polyphasic analysis involving phylogenetic, phylogenomic, physiological and chemotaxonomic analyses described in this study, GRR-S3-23T is considered to represent a novel species within the genus Tenacibaculum, for which the name Tenacibaculum tangerinum is proposed. The type strain is GRR-S3-23T (=KCTC 102029T=KACC 23271T=JCM 36353T).

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.

Roseobacter Group Probiotics Exhibit Differential Killing of Fish Pathogenic Tenacibaculum Species.

Fish-pathogenic bacteria of the Tenacibaculum genus are a serious emerging concern in modern aquaculture, causing tenacibaculosis in a broad selection of cultured finfish. Data describing their virulence mechanisms are scarce and few means, antibiotic treatment aside, are available to control their proliferation in aquaculture systems. We genome sequenced a collection of 19 putative Tenacibaculum isolates from outbreaks at two aquaculture facilities and tested their susceptibility to treatment with tropodithietic acid (TDA)-producing Roseobacter group probiotics. We found that local outbreaks of Tenacibaculum can involve heterogeneous assemblages of species and strains with the capacity to produce multiple different virulence factors related to host invasion and infection. The probiotic Phaeobacter piscinae S26 proved efficient in killing pathogenic Tenacibaculum species such as T. maritimum, T. soleae, and some T. discolor strains. However, the T. mesophilum and T. gallaicum species exhibit natural tolerance toward TDA and are hence not likely to be easily killed by TDA-producing probiotics. Tolerance toward TDA in Tenacibaculum is likely involving multiple inherent physiological features pertaining to electron and proton transport, iron sequestration, and potentially also drug efflux mechanisms, since genetic determinants encoding such features were significantly associated with TDA tolerance. Collectively, our results support the use of TDA producers to prevent tenacibaculosis; however, their efficacy is likely limited to some Tenacibaculum species. IMPORTANCE A productive and sustainable aquaculture sector is needed to meet the UN sustainable development goals and supply the growing world population with high-protein food sources. A sustainable way to prevent disease outbreaks in the industry is the application of probiotic bacteria that can antagonize fish pathogens in the aquaculture systems. TDA-producing Roseobacter group probiotics have proven efficient in killing important vibrio pathogens and protecting fish larvae against infection, and yet their efficacy against different fish pathogenic species of the Tenacibaculum genus has not been explored. Therefore, we tested the efficacy of such potential probiotics against a collection of different Tenacibaculum isolates and found the probiotic to efficiently kill a subset of relevant strains and species, supporting their use as sustainable disease control measure in aquaculture.

Development and evaluation of a rapid, specific, and sensitive loop-mediated isothermal amplification assay to detect Tenacibaculum sp. strain pbs-1 associated with black-spot shell disease in Akoya pearl oysters.

Black-spot shell disease decreases pearl quality and threatens pearl oyster survival. Establishment of a rapid, specific, and sensitive assay to detect Tenacibaculum sp. strain Pbs-1 associated with black-spot shell disease is of commercial importance. We developed a rapid, specific, and highly sensitive loop-mediated isothermal amplification (LAMP) assay to detect Tenacibaculum sp. Pbs-1 in Akoya pearl oysters Pinctada fucata. A set of five specific primers (two inner, two outer, and a loop) were designed based on the 16S-23S internal spacer region of strain Pbs-1. The optimum reaction temperature was 63 °C, and concentrations of the inner and loop primers were 1.4 and 1.0 µM, respectively. The LAMP product can be detected using agarose gel electrophoresis, and the color change in the reaction tube can be detected visually (by the naked eye) following the addition of malachite green. Our assay proved to be specific for strain Pbs-1, with no cross-reactivity with five other species of Tenacibaculum. The detection limit of the LAMP assay at 35 min is 50 pg, and at 60 min it is 5 fg. We evaluated the LAMP assay using diseased and healthy pearl oysters. The results demonstrate the suitability and simplicity of this test for rapid field diagnosis of strain Pbs-1.

Tenacibaculum aquimarinum sp. nov., isolated from a marine alga and seawater.

Two Gram-stain-negative, aerobic and yellow-pigmented bacterial strains, designated K20-16T and MSW2, were isolated from a marine red alga (Chondrus species) and seawater, respectively. Both strains were oxidase-positive, weakly catalase-positive and non-flagellated rods with gliding motility. Menaquinone-6 was detected as the sole isoprenoid quinone in both strains. Iso-C15:0, iso-C15:0 3-OH, iso-C15:1 G, C15:1 ω6c and summed feature 3 (comprising C16:1 ω7c and/or C16:1 ω6c) were identified in both strains as major fatty acids. Phosphatidylethanolamine was not identified in strain K20-16T, but it was identified in strain MSW2. The genomic DNA G+C contents of strains K20-16T and MSW2 were 30.5 and 30.7 %, respectively. Strains K20-16T and MSW2 shared 99.7% 16S rRNA gene sequence similarity, 97.7% average nucleotide identity (ANI), and 80.5% digital DNA-DNA hybridization (DDH) value, indicating that they are the same species. Phylogenetic analyses based on 16S rRNA gene and 92 concatenated core protein sequences revealed that strains K20-16T and MSW2 formed a phylogenic lineage within the genus Tenacibaculum and were most closely related to Tenacibaculum todarodis LPB0136T with 98.3 and 98.0% 16S rRNA gene sequence similarities, respectively. ANI and digital DDH values between strains K20-16T and MSW2 and other type strains were less than 91.4 and 43.1 %, respectively. Based on the phenotypic, chemotaxonomic and molecular features, strains K20-16T and MSW2 represent a novel species of the genus Tenacibaculum, for which the name Tenacibaculum aquimarinum sp. nov. is proposed. The type strain is K20-16T (=KACC 22 342T=JCM 35 023T).

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.

Seven New Alkaloids Isolated from Marine Flavobacterium Tenacibaculum discolor sv11.

Marine flavobacterium Tenacibaculum discolor sv11 has been proven to be a promising producer of bioactive nitrogen-containing heterocycles. A chemical investigation of T. discolor sv11 revealed seven new heterocycles, including the six new imidazolium-containing alkaloids discolins C-H (1−6) and one pyridinium-containing alkaloid dispyridine A (7). The molecular structure of each compound was elucidated by analysis of NMR and HR-ESI-MS data. Furthermore, enzymatic decarboxylation of tryptophan and tyrosine to tryptamine and tyramine catalyzed by the decarboxylase DisA was investigated using in vivo and in vitro experiments. The antimicrobial activity of the isolated compounds (1−7) was evaluated. Discolin C and E (1 and 3) exhibited moderate activity against Gram-positive Bacillus subtilis DSM10, Mycobacterium smegmatis ATCC607, Listeria monocytogenes DSM20600 and Staphylococcus aureus ATCC25923, with MIC values ranging from 4 µg/mL to 32 µg/mL.

Tenacibaculosis in Norwegian Atlantic salmon (Salmo salar) cage-farmed in cold sea water is primarily associated with Tenacibaculum finnmarkense genomovar finnmarkense.

Skin conditions associated with Tenacibaculum spp. constitute a significant threat to the health and welfare of sea-farmed Atlantic salmon (Salmo salar L.) in Norway. Fifteen presumptive tenacibaculosis outbreaks distributed along the Norwegian coast during the late winter and spring of 2018 were investigated. Bacteriological culture confirmed the presence of Tenacibaculum spp. Seventy-six isolates cultured from individual fish were selected and subjected to whole-genome sequencing and MALDI-TOF MS analysis. Average nucleotide identity and MALDI-TOF analyses confirmed the presence of T. finnmarkense and T. dicentrarchi, with further division of T. finnmarkense into genomovars (gv.) finnmarkense and ulcerans. Core genome multilocus sequence typing (cgMLST) and single-nucleotide polymorphism (SNP) analyses identified the presence of a genetically conserved cluster of gv. finnmarkense isolates against a background of relatively genetically diverse gv. finnmarkense and gv. ulcerans isolates in 13 of the 15 studied cases. This clustering strongly suggests a link between T. finnmarkense gv. finnmarkense and development of clinical tenacibaculosis in sea-farmed Norwegian salmon in the late winter and spring. Analysis of 25 Tenacibaculum isolates collected during the spring of 2019 from similar cases identified a similar distribution of genotypes. Low water temperatures were common to all cases, and most incidences involved relatively small fish shortly after sea transfer, suggesting that these fish are particularly predisposed to Tenacibaculum infection.

Tenacibactins K-M, cytotoxic siderophores from a coral-associated gliding bacterium of the genus Tenacibaculum.

HPLC/DAD-based chemical investigation of a coral-associated gliding bacterium of the genus Tenacibaculum yielded three desferrioxamine-class siderophores, designated tenacibactins K (1), L (2), and M (3). Their chemical structures, comprising repeated cadaverine-succinic acid motifs terminated by a hydroxamic acid functionality, were elucidated by NMR and negative MS/MS experiments. Compounds 1-3 were inactive against bacteria and a yeast but displayed cytotoxicity against 3Y1 rat embryonic fibroblasts and P388 murine leukemia cells at GI50 in submicromolar to micromolar ranges. Their iron-chelating activity was comparable to deferoxamine mesylate.

Tenacibaculum pelagium sp. nov., isolated from marine sediment.

A novel bright yellow pigmented, Gram-stain-negative, gliding, aerobic and rod-shaped marine bacterium, designated strain S7007T, was isolated from a marine sediment sample taken from Jingzi Wharf, Weihai, China. The bacterium was able to grow at 4-33 °C (optimum 28 °C), at pH 6.5-9.0 (optimum 7.0) and with 2.0-4.0% (w/v) NaCl (optimum 3.0%). According to the phylogenetic analysis based on the 16S rRNA gene sequences, strain S7007T was associated with the genus Tenacibaculum and showed highest similarity to Tenacibaculum adriaticum JCM 14633T (98.0%). The average nucleotide identity (ANI) scores of strain S7007T with T. adriaticum JCM 14633T and T. maritimum NBRC 110778T were 78.3% and 77.1%, respectively and the Genome-to-Genome Distance Calculator (dDDH) scores were 20.5% and 19.9%, respectively. The sole isoprenoid quinone was MK-6 and the major cellular fatty acids (> 10.0%) were iso-C15:0, iso-C15:0 3-OH, iso-C15: 1 G and summed feature 3 (comprising C16:1 ω7c and/or C16:1 ω6c). The major polar lipids of strain S7007T were phosphatidylethanolamine, phosphatidyldimethylethanolamine, one unidentified lipid and two unidentified aminolipids. The genomic DNA G + C content was 30.9 mol %. The combined phenotypic data and phylogenetic inference that strain S7007T should be classified as a novel species in the genus Tenacibaculum, for which the name Tenacibaculum pelagium sp. nov. is proposed. The type strain is S7007T (= MCCC 1H00428T = KCTC 72941T).

Tenacibaculum maritimum CRISPR loci analysis and evaluation of isolate spoligotyping.

AIMS: We performed in silico analysis of CRISPRcas loci from Tenacibaculum maritimum, evaluated spoligotyping as a subtyping method and genotyped uncharacterized Turkish isolates from European sea bass by multilocus sequence typing (MLST). METHODS AND RESULTS: Spoligotyping was performed with primers designed to allow amplification and sequencing of whole CRISPR-arrays from 23 T. maritimum isolates. Twenty-three completed/draft genomes were also downloaded from the NCBI database and analysed. MLST of Turkish isolates was achieved with a well-established 7-gene scheme. Tenacibaculum maritimum genomes carry a structurally complete but partially defective class II CRISPRcas locus due to known amino acid substitutions in encoded Cas9 proteins. Our spacer identification suggests that the host range of bacteriophage P2559Y and Vibrio phage nt-1 include T. maritimum and that the most recurrent infection recorded by isolates has been with Tenacibaculum phage PTm5. Thirty-eight isolates with this CRISPRcas locus belonged to 25 spoligotypes and to 24 sequence types by MLST, respectively. According to MLST, T. maritimum isolates from Turkey are most related to previously defined sequence types ST3, ST40 and ST41 isolates from Spain, Malta and France. CONCLUSIONS: The evaluated spoligotyping offers discriminatory power comparable to MLST. SIGNIFICANCE AND IMPACT OF THE STUDY: Spoligotyping has potential as a quick, easy and cheap tool for subtyping of T. maritimum isolates.

Quantitative PCR for Tenacibaculum dicentrarchi and T. finnmarkense.

Numerous Tenacibaculum species, including T. dicentrarchi, T. maritimum and T. finnmarkense, are associated with tenacibaculosis in finfish; however, quantitative identification techniques are limited. Quantitative PCR assays were developed to detect T. dicentrarchi and T. finnmarkense. TaqMan assays using 16S rDNA demonstrated low detection limits (0.07-269 bacteria), suitable amplification efficiencies (>86%) and moderate specificity. However, the amplification of isolates with 100% sequence similarity to T. finnmarkense AY7486TD using both the T. finnmarkense and T. dicentrarchi assays indicates that other genes should be investigated. Both assays may help describe the pathogenesis of tenacibaculosis and may aid management practices for the aquaculture 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.

Tenacibaculum piscium sp. nov., isolated from skin ulcers of sea-farmed fish, and description of Tenacibaculum finnmarkense sp. nov. with subdivision into genomovars finnmarkense and ulcerans.

Results of previous multilocus sequence and whole-genome-based analyses have suggested that a homogeneous group of isolates belonging to the genus Tenacibaculum, represented by strain TNO020T and associated with skin ulcer development in sea-farmed fish, represents an as-yet-undescribed species. Comparative whole-genome analysis performed in the present study clustered five isolates, including TNO020T, in a distinct lineage within the genus Tenacibaculum. Phenotypic differences, high intra-cluster average nucleotide identity (ANI) values and low ANI values with other Tenacibaculum species support the proposal of a novel species, for which we propose the name Tenacibaculum piscium sp. nov. with strain TNO020T (=CCUG 73833T=NCIMB 15240T) as the type strain. Further, large-scale genome analyses confirmed the existence of two different phylogenetic lineages within 'T. finnmarkense', a species effectively but not validly published previously. ANI values just above the species delineation threshold of 95-96 % confirmed that both lineages belong to the same species. This result was also supported by DNA-DNA hybridization values. Phenotypically, the two conspecific lineages are distinguishable by differences in growth temperature range and ability to degrade l-proline. For the group of isolates already commonly known as 'T. finnmarkense', we propose the name Tenacibaculum finnmarkense sp. nov., with strain TNO006T (=CCUG 73831T=NCIMB 15238T) as the type strain. We further propose the subdivision of T. finnmarkense sp. nov. into two genomovars, T. finnmarkense genomovar finnmarkense with strain TNO006T (=CCUG 73831T=NCIMB 15238T) as the type strain and T. finnmarkense genomovar ulcerans with strain TNO010T (=CCUG 73832T=NCIMB 15239T) as the type strain.

Mucosal immune responses in Senegalese sole (Solea senegalensis) juveniles after Tenacibaculum maritimum challenge: A comparative study between ocular and blind sides.

Most pathogens start the process of infection at the mucosal surfaces and therefore the mucosal immune response plays an essential role in the course of the infection. Due to the Senegalese sole (Solea senegalensis Kaup) condition of flatfish, the present comparative study aimed to analyse several immune-related enzymes as well as the bactericidal activity in the skin mucus from ocular and blind sides. For this purpose, Senegalese sole juveniles were bath challenged with a sub-lethal dose of Tenacibaculum maritimum for 24 h and sampled at 1, 2 and 3 weeks. The haematological profile and immune-related parameters were also measured in plasma in order to evaluate the systemic immune response after T. maritimum challenge. Results from this study showed that most parameters tested increased in skin mucus of bath challenged fish compared to unchallenged ones. In contrast, the sub-lethal dose tested did not influence the haematological profile including peripheral numbers the different leucocyte types. No variations were observed in plasma lysozyme, peroxidase, protease and haemolytic complement activities between unchallenged and bath challenged fish. This study suggests that the studied innate immune-related molecules are constitutively present in both skin mucus sides but at different levels. Interestingly, the levels of most parameters measured were higher on the ocular side than on the blind side, possibly due to the higher exposure to invasion by waterborne microorganisms on this side. Therefore, the present study brings some insights regarding local immune responses after bacterial challenge in skin mucus from the ocular and blind sides in one of the most valuable flatfish species in southern Europe.