Draft genome for Flavobacterium psychrophilum isolates from diseased coho salmon (Oncorhynchus kisutch) in Chile.

We present the draft genome sequences of six Flavobacterium psychrophilum isolates recovered from diseased coho salmon (Oncorhynchus kisutch) cultured by two farms in Chile. This study provides the first detailed insights into the genomic characteristics of this fish pathogen recovered from a host with limited information and cultured in Chile.

Estimating taxonomic and functional structure along a tropical estuary: linking metabolic traits and aspects of ecosystem functioning.

Microbial life forms are among the most ubiquitous on Earth, yet many remain understudied in Caribbean estuaries. We report on the prokaryote community composition of the Urabá Estuary in the Colombian Caribbean using 16S rRNA gene-transcript sequencing. We also assessed potential functional diversity through 38 metabolic traits inferred from 16S rRNA gene data. Water samples were collected from six sampling stations at two depths with contrasting light-penetration conditions along an approximately 100 km transect in the Gulf of Urabá in December 2019. Non-metric multidimensional scaling analysis grouped the samples into two distinct clusters along the transect and between depths. The primary variables influencing the prokaryote community composition were the sampling station, depth, salinity, and dissolved oxygen levels. Twenty percent of genera (i.e., 58 out 285) account for 95% of the differences between groups along the transect and among depths. All of the 38 metabolic traits studied showed some significant relationship with the tested environmental variables, especially salinity and except with temperature. Another non-metric multidimensional scaling analysis, based on community-weighted mean of traits, also grouped the samples in two clusters along the transect and over depth. Biodiversity facets, such as richness, evenness, and redundancy, indicated that environmental variations-stemming from river discharges-introduce an imbalance in functional diversity between surface prokaryote communities closer to the estuary's head and bottom communities closer to the ocean. Our research broadens the use of 16S rRNA gene transcripts beyond mere taxonomic assignments, furthering the field of trait-based prokaryote community ecology in transitional aquatic ecosystems.IMPORTANCEThe resilience of a dynamic ecosystem is directly tied to the ability of its microbes to navigate environmental gradients. This study delves into the changes in prokaryote community composition and functional diversity within the Urabá Estuary (Colombian Caribbean) for the first time. We integrate data from 16S rRNA gene transcripts (taxonomic and functional) with environmental variability to gain an understanding of this under-researched ecosystem using a multi-faceted macroecological framework. We found that significant shifts in prokaryote composition and in primary changes in functional diversity were influenced by physical-chemical fluctuations across the estuary's environmental gradient. Furthermore, we identified a potential disparity in functional diversity. Near-surface communities closer to the estuary's head exhibited differences compared to deeper communities situated farther away. Our research serves as a roadmap for posing new inquiries about the potential functional diversity of prokaryote communities in highly dynamic ecosystems, pushing forward the domain of multi-trait-based prokaryote community ecology.

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

Strain T-12T, an orange, Gram-stain-negative, non-motile, rod-shaped strain, was isolated in November 2013 from water samples collected from an Atlantic salmon (Salmo salar) fry culturing system at a fish farm in Chile. Phylogenetic analysis based on 16S rRNA sequences (1394 bp) revealed that strain T-12T belonged to the genus Flavobacterium, showing close relationships to Flavobacterium bernardetii F-372T (99.48 %) and Flavobacterium terrigena DS-20T (98.50 %). The genome size of strain T-12T was 3.28 Mb, with a G+C content of 31.1 mol%. Genome comparisons aligned strain T-12T with Flavobacterium bernardetii F-372T (GCA_011305415) and Flavobacterium terrigena DSM 17934T (GCA_900108955). The highest digital DNA-DNA hybridization (dDDH) values were 42.6 % with F. bernardetii F-372T (GCA_011305415) and 33.9 % with F. terrigena DSM 17934T (GCA_900108955). Pairwise average nucleotide identity (ANI) calculations were below the species cutoff, with the best results with F. bernardetii F-372T being: ANIb, 90.33 %; ANIm, 91.85 %; and TETRA, 0.997 %. These dDDH and ANI results confirm that strain T-12T represents a new species. The major fatty acids were iso-C15 : 0 and C15 : 1ω6с. Detected polar lipids included phospholipids (n=2), aminophospholipid (n=1), aminolipid (n=1) and unidentified lipids (n=2). The predominant respiratory quinone was menaquinone MK7 (80 %) followed by MK-6 (20 %). Phenotypic, chemotaxonomic, and genomic data support the classification of strain T-12T (=CECT 30410T=RGM 3222T) as representing a novel species of Flavobacterium, for which the name Flavobacterium facile sp. nov. is proposed.

Discovery and Characterization of the ddx41 Gene in Atlantic Salmon: Evolutionary Implications, Structural Functions, and Innate Immune Responses to Piscirickettsia salmonis and Renibacterium salmoninarum Infections.

The innate immune response in Salmo salar, mediated by pattern recognition receptors (PRRs), is crucial for defending against pathogens. This study examined DDX41 protein functions as a cytosolic/nuclear sensor for cyclic dinucleotides, RNA, and DNA from invasive intracellular bacteria. The investigation determined the existence, conservation, and functional expression of the ddx41 gene in S. salar. In silico predictions and experimental validations identified a single ddx41 gene on chromosome 5 in S. salar, showing 83.92% homology with its human counterpart. Transcriptomic analysis in salmon head kidney confirmed gene transcriptional integrity. Proteomic identification through mass spectrometry characterized three unique peptides with 99.99% statistical confidence. Phylogenetic analysis demonstrated significant evolutionary conservation across species. Functional gene expression analysis in SHK-1 cells infected by Piscirickettsia salmonis and Renibacterium salmoninarum indicated significant upregulation of DDX41, correlated with increased proinflammatory cytokine levels and activation of irf3 and interferon signaling pathways. In vivo studies corroborated DDX41 activation in immune responses, particularly when S. salar was challenged with P. salmonis, underscoring its potential in enhancing disease resistance. This is the first study to identify the DDX41 pathway as a key component in S. salar innate immune response to invading pathogens, establishing a basis for future research in salmonid disease resistance.

Proteomic characterization of Tenacibaculum dicentrarchi under iron limitation reveals an upregulation of proteins related to iron oxidation and reduction metabolism, iron uptake systems and gliding motility.

A strategy for vaccine design involves identifying proteins that could be involved in pathogen-host interactions. The aim of this proteomic study was to determine how iron limitation affects the protein expression of Tenacibaculum dicentrarchi, with a primary focus on virulence factors and proteins associated with iron uptake. The proteomic analysis was carried out using two strains of T. dicentrarchi grown under normal (control) and iron-limited conditions, mimicking the host environment. Our findings revealed differences in the proteins expressed by the type strain CECT 7612T and the Chilean strain TdCh05 of T. dicentrarchi. Nonetheless, both share a common response to iron deprivation, with an increased expression of proteins associated with iron oxidation and reduction metabolism (e.g., SufA, YpmQ, SufD), siderophore transport (e.g., ExbD, TonB-dependent receptor, HbpA), heme compound biosynthesis, and iron transporters under iron limitation. Proteins involved in gliding motility, such as GldL and SprE, were also upregulated in both strains. A negative differential regulation of metabolic proteins, particularly those associated with amino acid biosynthesis, was observed under iron limitation, reflecting the impact of iron availability on bacterial metabolism. Additionally, the TdCh05 strain exhibited unique proteins associated with gliding motility machinery and phage infection control compared to the type strain. These groups of proteins have been identified as virulence factors within the Flavobacteriaceae family, including the genus Tenacibaculum. These results build upon our previous report on iron acquisition mechanisms and could lay the groundwork for future studies aimed at elucidating the role of some of the described proteins in the infectious process of tenacibaculosis, as well as in the development of potential vaccines.

Characterization of Tenacibaculum maritimum isolated from diseased salmonids farmed in Chile reveals high serological and genetic heterogeneity.

The diversity of Tenacibaculum maritimum in Chile remains poorly understood, particularly in terms of antigenic and genetic diversity. This information is crucial for the future development of a vaccine against tenacibaculosis and would increase understanding of this important fish pathogen. With this aim, the biochemical, antigenic, and genetic characteristics were analysed for 14 T. maritimum isolates, recovered from diseased Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss) farmed in Chile between 1998 and 2022. Biochemical analysis showed a homogeneity among all the Chilean T. maritimum isolates and all four other strains included for comparison purposes. Serological characterization using dot-blot assaying revealed antigenic heterogeneity with the use of unabsorbed antisera. The majority of isolates showed cross-reactions, identifying three main serological patterns. When the PCR-based serotyping scheme was performed, the existence of antigenic heterogeneity was confirmed. Four Atlantic salmon isolates were 4-0; and most isolates, including the rainbow trout isolate, were 3-1 (n = 9). A turbot (Scophthalmus maximus) isolate was 1-0. Using an existing Multilocus Sequence Typing system, two newly identified sequence types (ST193 and ST198) in the database were detected. ST193 encompassed nine isolates obtained from Atlantic salmon and rainbow trout, while ST198 regrouped four isolates, all retrieved from diseased Atlantic salmon in 2022. These findings highlight significant antigenic and genetic diversity among the Chilean isolates. This information is useful for epizootiology and the selection of suitable candidate strain(s) for vaccine development against tenacibaculosis caused by T. maritimum in Chilean salmon farming.

Unveiling Novel Kunitz- and Waprin-Type Toxins in the Micrurus mipartitus Coral Snake Venom Gland: An In Silico Transcriptome Analysis.

Kunitz-type peptide expression has been described in the venom of snakes of the Viperidae, Elapidae and Colubridae families. This work aimed to identify these peptides in the venom gland transcriptome of the coral snake Micrurus mipartitus. Transcriptomic analysis revealed a high diversity of venom-associated Kunitz serine protease inhibitor proteins (KSPIs). A total of eight copies of KSPIs were predicted and grouped into four distinctive types, including short KSPI, long KSPI, Kunitz-Waprin (Ku-WAP) proteins, and a multi-domain Kunitz-type protein. From these, one short KSPI showed high identity with Micrurus tener and Austrelaps superbus. The long KSPI group exhibited similarity within the Micrurus genus and showed homology with various elapid snakes and even with the colubrid Pantherophis guttatus. A third group suggested the presence of Kunitz domains in addition to a whey-acidic-protein-type four-disulfide core domain. Finally, the fourth group corresponded to a transcript copy with a putative 511 amino acid protein, formerly annotated as KSPI, which UniProt classified as SPINT1. In conclusion, this study showed the diversity of Kunitz-type proteins expressed in the venom gland transcriptome of M. mipartitus.

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 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.

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.

Method for lineage typing of epidemic Renibacterium salmoninarum in Chilean salmon farms.

Renibacterium salmoninarum (Rs) is the etiological agent of bacterial kidney disease (BKD), which significantly affects farmed and wild salmonids worldwide. Although the whole genome of Rs (~3.1 million nucleotides) is highly conserved, genomic epidemiology analyses have identified four sub-lineages from Chilean isolates. A total of 94 Rs genomes from the BIGSdb aquaculture database were aligned and compared using bioinformatics tools, identifying 2199 independent single-nucleotide polymorphisms (SNPs) spread along the genome. A detailed analysis of the distribution of the SNPs showed five local zones of a length in the range of 10-15 kbp that should be used to unambiguously identify a specific sub-lineage. Based on the Rs type strain DSM 20767T , we designed multiplex PCR primers that produce specific amplification products which were further sequenced by the Sanger method to obtain the genotype of the sub-lineage. For the genetic typing, we evaluated 27 Rs isolates recovered from BKD outbreaks from different fish species and regions of Chile. Based on the findings reported here, we propose the PCR approach as a valuable tool for the rapid and reliable studying of the relationships between Rs isolates and the different sub-lineages without requiring the sequencing of the entire genome.

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.