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.

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.

Comparative genomic assessment of members of genus Tenacibaculum: an exploratory study.

Tenacibaculosis is an ulcerative skin disorder that affects finfish. It is caused by members of the genus Tenacibaculum, resulting in eccentric behavioural changes, including anorexia, lethargy, and abnormal swimming patterns that often result in mortality. Currently, species suspected of causing fish mortality include T. ovolyticum, T. gallaicum, T. discolor, T. finnmarkense, T. mesophilum, T. soleae, T. dicentrarchi, and T. maritimum. However, pathogenic members and the mechanisms involved in disease causation, progression, and transmission are limited due to the inadequate sequencing efforts in the past decade. In this study, we use a comparative genomics approach to investigate the characteristic features of 26 publicly available genomes of Tenacibaculum and report our observations. We propose the reclassification of "T. litoreum HSC 22" to the singaporense species and assignment of "T. sp. 4G03" to the species discolor (species with quotation marks have not been appropriately named). We also report the co-occurrence of several antimicrobial resistance/virulence genes and genes private to a few members. Finally, we mine several non-B DNA forming regions, operons, tandem repeats, high-confidence putative effector proteins, and sortase that might play a pivotal role in bacterial evolution, transcription, and pathogenesis.

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.

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.

Genomic characterization of Tenacibaculum maritimum O-antigen gene cluster and development of a multiplex PCR-based serotyping scheme.

Tenacibaculum maritimum is a devastating bacterial pathogen affecting a large variety of marine fish species. It is responsible for significant economic losses in aquaculture farms worldwide. Different typing methods have been proposed to analyse bacterial diversity and population structure. Serological heterogeneity has been observed and up to four different serotypes have been described so far. However, the underlying molecular factors remain unknown. By combining conventional serotyping and genome-wide association study, we identified the genomic loci likely involved in the O-antigen biosynthesis. This finding allowed the development of a robust multiplex PCR-based serotyping scheme able to detect subgroups within each serotype and therefore performs better than conventional serotyping. This scheme was successfully applied to a large number of isolates from worldwide origin and retrieved from a large variety of fish species. No obvious correlations were observed between the mPCR-based serotype and the host species or the geographic origin of the isolates. Strikingly, the distribution of mPCR-based serotypes does not follow the core genome phylogeny. Nevertheless, this simple and cost-effective mPCR-based serotyping method could be useful for different applications such as population structure analysis, disease surveillance, vaccine formulation and efficacy follow-up.

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.

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.

Tenacibaculosis caused by Tenacibaculum maritimum: Updated knowledge of this marine bacterial fish pathogen.

Tenacibaculosis occurs due to the marine bacterial pathogen Tenacibaculum maritimum. This ulcerative disease causes high mortalities for various marine fish species worldwide. Several external clinical signs can arise, including mouth erosion, epidermal ulcers, fin necrosis, and tail rot. Research in the last 15 years has advanced knowledge on the traits and pathogenesis mechanisms of T. maritimum. Consequently, significant progress has been made in defining the complex host-pathogen relationship. Nevertheless, tenacibaculosis pathogenesis is not yet fully understood. Continued research is urgently needed, as demonstrated by recent reports on the re-emerging nature of tenacibaculosis in salmon farms globally. Current sanitary conditions compromise the development of effective alternatives to antibiotics, in addition to hindering potential preventive measures against tenacibaculosis. The present review compiles knowledge of T. maritimum reported after the 2006 review by Avendaño-Herrera and colleagues. Essential aspects are emphasized, including antigenic and genomic characterizations and molecular diagnostic procedures. Further summarized are the epidemiological foundations of the T. maritimum population structure and elucidations as to the virulence mechanisms of pathogenic isolates, as found using biological, microbiological, and genomic techniques. This comprehensive source of reference will undoubtable serve in tenacibaculosis prevention and control within the marine fish farming industry. Lastly, knowledge gaps and valuable research areas are indicated as potential guidance for future studies.

Phylogenetic analyses of Norwegian Tenacibaculum strains confirm high bacterial diversity and suggest circulation of ubiquitous virulent strains.

Tenacibaculosis is a bacterial ulcerative disease affecting marine fish and represents a major threat to aquaculture worldwide. Its aetiological agents, bacteria belonging to the genus Tenacibaculum, have been present in Norway since at least the late 1980's and lead to regular ulcerative outbreaks and high mortalities in production of farmed salmonids. Studies have shown the presence of several Tenacibaculum species in Norway and a lack of clonality in outbreak-related strains, thus preventing the development of an effective vaccine. Hence, a thorough examination of the bacterial diversity in farmed fish presenting ulcers and the geographical distribution of the pathogens should provide important insights needed to strengthen preventive actions. In this study, we investigated the diversity of Tenacibaculum strains isolated in 28 outbreaks that occurred in Norwegian fish farms in the period 2017-2020. We found that 95% of the 66 strains isolated and characterized, using an existing MultiLocus Sequence Typing system, have not previously been identified, confirming the high diversity of this genus of bacteria in Norway. Several of these Tenacibaculum species seem to be present within restricted areas (e.g., Tenacibaculum dicentrarchi in western Norway), but phylogenetic analysis reveals that several of the strains responsible of ulcerative outbreaks were isolated from different localities (e.g., ST- 172 isolated from northern to southern parts of Norway) and/or from different hosts. Understanding their reservoirs and transmission pathways could help to address major challenges in connection with prophylactic measures and development of vaccines.

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.

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.

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).

New salmonid hosts for Tenacibaculum species: Expansion of tenacibaculosis in Chilean aquaculture.

The success and sustainability of Chilean aquaculture largely depends on the control of endemic and emerging pathogens, including several species of the genus Tenacibaculum. Tenacibaculum dicentrarchi and "Tenacibaculum finnmarkense" have been detected and confirmed in Chilean Atlantic salmon (Salmo salar). However, no outbreaks of tenacibaculosis in rainbow trout (Oncorhynchus mykiss) or coho salmon (Oncorhynchus kisutch) have been reported, either in Chile or globally. The aims of this study were to determine whether the mortalities recorded for rainbow trout and coho salmon from five marine fish farms located in the Los Lagos, Aysén, and Magallanes Regions could be caused by Tenacibaculum spp. The diseased fish exhibited cutaneous haemorrhages, tail and peduncle rots, and damage on the mouth and tongue. Microbiological analysis of infected external tissues yielded 13 bacterial isolates. The isolates were identified as members of the genus Tenacibaculum through biochemical analysis (e.g. Gram-stain negative, straight rods, filamentous cells and motile by gliding), but differences existed in biochemical results, making species-level identification through biomolecular tools essential. The 16S rRNA analysis found that the majority of isolates were more closely related to "T. finnmarkense" than T. dicentrarchi, while the phylogenetic trees resulting from multilocus sequence data recovered the four main clades (clades I to IV) identified by Olsen et al. (2017, Veterinary Microbiology, 205, 39). This is the first documented occurrence of clinical tenacibaculosis in farmed rainbow trout and coho salmon globally, and it extends the known host distribution of this pathogen in Chile. Moreover, we confirm the presence of Tenacibaculum species in the Chilean Patagonia. These findings highlight the importance of establishing preventative measures to minimize the spread of this disease within the Chilean marine aquaculture industry, as well as the need for monitoring initiatives worldwide in these farmed fish species.

Genetic diversity and population structure of Tenacibaculum maritimum, a serious bacterial pathogen of marine fish: from genome comparisons to high throughput MALDI-TOF typing.

Tenacibaculum maritimum is responsible for tenacibaculosis, a devastating marine fish disease. This filamentous bacterium displays a very broad host range and a worldwide geographical distribution. We analyzed and compared the genomes of 25 T. maritimum strains, including 22 newly draft-sequenced genomes from isolates selected based on available MLST data, geographical origin and host fish. The genome size (~3.356 Mb in average) of all strains is very similar. The core genome is composed of 2116 protein-coding genes accounting for ~75% of the genes in each genome. These conserved regions harbor a moderate level of nucleotide diversity (~0.0071 bp-1) whose analysis reveals an important contribution of recombination (r/m ≥ 7) in the evolutionary process of this cohesive species that appears subdivided into several subgroups. Association trends between these subgroups and specific geographical origin or ecological niche remains to be clarified. We also evaluated the potential of MALDI-TOF-MS to assess the variability between T. maritimum isolates. Using genome sequence data, several detected mass peaks were assigned to ribosomal proteins. Additionally, variations corresponding to single or multiple amino acid changes in several ribosomal proteins explaining the detected mass shifts were identified. By combining nine polymorphic biomarker ions, we identified combinations referred to as MALDI-Types (MTs). By investigating 131 bacterial isolates retrieved from a variety of isolation sources, we identified twenty MALDI-Types as well as four MALDI-Groups (MGs). We propose this MALDI-TOF-MS Multi Peak Shift Typing scheme as a cheap, fast and an accurate method for screening T. maritimum isolates for large-scale epidemiological surveys.

Insights into the microbiome of farmed Asian sea bass (Lates calcarifer) with symptoms of tenacibaculosis and description of Tenacibaculum singaporense sp. nov.

Outbreaks of diseases in farmed fish remain a recurring problem despite the development of vaccines and improved hygiene standards on aquaculture farms. One commonly observed bacterial disease in tropical aquaculture of the South-East Asian region is tenacibaculosis, which is attributed to members of the genus Tenacibaculum (family Flavobacteriaceae, phylum Bacteroidetes), most notably Tenacibaculum maritimum. The impact of tenacibaculosis on the fish microbiota remains poorly understood. In this study, we analysed the microbiota of different tissues of commercially reared Asian seabass (Lates calcarifer) that showed symptoms of tenacibaculosis and compared the microbial communities to those of healthy and experimentally infected fish that were exposed to diseased farmed fish. The relative abundance of Tenacibaculum species in experimentally infected fish was significantly lower than in commercially reared diseased fish and revealed a higher prevalence of different Tenacibaculum species. One isolated strain, TLL-A2T, shares 98.7% 16S rRNA gene identity with Tenacibaculum mesophilum DSM 13764T. The genome of strain TLL-A2T was sequenced and compared to that of T. mesophilum DSM 13764T. Analysis of average nucleotide identity and comparative genome analysis revealed only 92% identity between T. mesophilum DSM 13764T and strain TLL-A2T and differences between the two strains in predicted carbohydrate activating enzymes respectively. Phenotypic comparison between strain TLL-A2T and T. mesophilum DSM 13764T indicated additional differences, such as growth response at different salt concentrations. Based on molecular and phenotypic differences, strain TLL-A2T (=DSM 106434T, KCTC 62393T) is proposed as the type strain of Tenacibaculum singaporense sp. nov.

A novel jumbo Tenacibaculum maritimum lytic phage with head-fiber-like appendages.

A novel jumbo bacteriophage (myovirus) is described. The lytic phage of Tenacibaculum maritimum, which is the etiological agent of tenacibaculosis in a variety of farmed marine fish worldwide, was plaque-isolated from seawater around a fish aquaculture field in Japan. The phage had an isometric head 110-120 nm in diameter, from which several 50- to 100-nm-long flexible fiber-like appendages emanate, and a 150-nm-long rigid contractile tail. The full genomes of the two representative phages (PTm1 and PTm5) were 224,680 and 226,876 bp long, respectively, both with 29.7% GC content, and the number of predicted open reading frames (ORFs) was 308 and 306, respectively. The average nucleotide sequence identity between PTm1 and PTm5 was 99.95%, indicating they are quite similar to each other. A genetic relationship was found in 15.0-16.6% of the predicted ORFs among the T. maritimum phages PTm1 and PTm5, the Tenacibaculum spp. phage pT24, and the Sphingomonas paucimobilis phage PAU. Phylogenetic analysis based on the terminase large subunit genes revealed that these four phages (PTm1, PTm5, pT24 and PAU) are more closely related than the other 10 jumbo myoviruses that have similar genome sizes. Transmission electron microscopy observations suggest that the head fibers of the T. maritimum phage function as tentacles to search and recognize the host cell surface to facilitate infection.

An outbreak of acute disease and mortality in Atlantic salmon (Salmo salar) post-smolts in Norway caused by Tenacibaculum dicentrarchi.

An outbreak of disease characterized by skin ulcers, fin rot and mortality was observed a few days after the transfer of Atlantic salmon (Salmo salar) from a freshwater smolt production facility to a land-based seawater post-smolt site. Dead and moribund fish had severe skin and muscle ulcers, often 2-6 cm wide, particularly caudal to the pectoral fins. Microscopic examination of smears from ulcers and head kidney identified long, slender Gram-negative rods. Histopathological analysis revealed abundance of long, slender Tenacibaculum-like bacteria in ulcers and affected fins. Genetic characterization using multilocus sequence analysis (MLSA) of seven housekeeping genes, including atpA, dnaK, glyA, gyrB, infB, rlmN and tgt, revealed that the isolates obtained during the outbreak were all clustered with the Tenacibaculum dicentrarchi-type strain (USC39/09T ) from Spain. Two bath challenge experiments with Atlantic salmon and an isolate of T. dicentrarchi from the outbreak were performed. No disease or mortality was observed in the first trial. In the second trial with a higher challenge dose of T. dicentrarchi and longer challenge time, we got 100% mortality within 48 hr. This is the first reported outbreak of disease caused by T. dicentrarchi in Norwegian farmed Atlantic salmon.

Optimisation and validation of a PCR to detect viable Tenacibaculum maritimum in salmon skin tissue samples.

A PCR protocol was optimised and validated for the detection of viable Tenacibaculum maritimum cells in salmon skin tissue. Viability conventional (vPCR) and quantitative PCR (v-qPCR) assays both had a limit of detection of 103 CFU mL-1 viable cells. The v-qPCR assay showed a linear quantification over 4 log units. Conventional vPCR showed complete signal suppression when only dead cells were present at concentrations lower than 106 CFU mL-1. While the v-qPCR did not result in complete suppression when only dead cells were present, a method was developed to determine if viable cells were present based on the % Δ in cycle threshold (Ct) value. The procedure was validated for high-throughput processing and an enrichment protocol was validated to reliably detect low concentrations of viable cells both with and without a high background of dead cells. Performing this protocol on naturally infected tissues showed that vPCR and v-qPCR reduced the potential for false positives compared to using conventional PCR and qPCR. The optimised protocol developed for this study provides an efficient, reliable and robust alternative for the detection of viable T. maritimum in skin tissue.

First identification in Tasmania of fish pathogens Tenacibaculum dicentrarchi and T. soleae and multiplex PCR for these organisms and T. maritimum.

This work describes a new polymerase chain reaction (PCR) assay for rapid identification of the fish pathogens Tenacibaculum dicentrarchi, T. maritimum and T. soleae, 3 organisms which can cause tenacibaculosis in farmed salmonids. The selected primers amplified a 688 bp fragment for T. dicentrarchi, a 288 bp fragment of the T. maritimum and a 183 bp fragment of the T. soleae 16S rRNA genes. The PCR assay was shown to be both specific and sensitive with a detection limit of approximately 50 fg DNA for each species in the presence of competing DNA. The multiplex PCR allowed detection of each pathogen from pure or mixed cultures, where the different Tenacibaculum species can be difficult to distinguish phenotypically. Our results indicate that the specific primers and PCR method developed here provide sensitive and fast detection of T. dicentrarchi, T. maritimum and T. soleae alone or in combination.