Inhibitors of LAMP used to detect Tenacibaculum sp. strain Pbs-1 associated with black-spot shell disease in Akoya pearl oysters, and additives to reduce the effect of the inhibitors.

Black-spot shell disease is an unresolved disease that decreases pearl quality and threatens pearl oyster survival. In previous studies, the bacterium Tenacibaculum sp. strain Pbs-1 was isolated from diseased Akoya pearl oysters Pinctada fucata, and a rapid, specific, and sensitive loop-mediated isothermal amplification (LAMP) assay for detecting this pathogen was established. This technology has considerable potential for routine diagnosis of strain Pbs-1 in oyster hatcheries and/or pearl farms; therefore, it is vital to identify substances in environmental samples that might inhibit LAMP and to find additives that can reduce the inhibition. In this study, we investigated the effects of six chemicals or proteins, otherwise known as conventional PCR inhibitors, on LAMP, using the DNA of strain Pbs-1 as template: humic acid, urea, iron (III) chloride hexahydrate, melanin, myoglobin, and Ethylenediamine-N,N,N',N'-tetraacetic acid, disodium salt, dihydrate (EDTA; pH 6.5). Next, to reduce the effects of identified inhibitors, we tested the addition of bovine serum albumin (BSA) or T4 gene 32 protein (gp32) to the LAMP assay. When 50 ng of DNA template was used, 4 ng/µL of humic acid, 0.05% melanin, and 10 mM of EDTA (pH 6.5) inhibited the LAMP reaction, whereas myoglobin, urea, and FeCl3 had no effect. When 50 pg of DNA template was used, 4 ng/µL of humic acid, 0.05% melanin, 4 µg/µL of myoglobin, 10 µg/µL of urea, and 10 mM of EDTA inhibited the LAMP reaction. Thus, it was shown that the gene-amplification inhibitory effect of melanin, humic acid, and urea could be reduced by adding BSA or gp32 to the LAMP reaction mixture. This technique could be applied as part of a protocol to prevent mass mortalities of pearl oysters; moreover, the results enhance our knowledge about substances that inhibit LAMP and methods to reduce the inhibition, which have rarely been reported.

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.

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.

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 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.

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.

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.

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.

Identification and typing of fish pathogenic species of the genus Tenacibaculum.

Tenacibaculosis is a major bacterial disease that causes severe fish outbreaks and losses and limits the culture of a variety of commercially valuable anadromous and marine fish species in Europe, America, Asia and Oceania. Fish affected by tenacibaculosis have external lesions and necrosis that affect different areas of the body surface, reducing their commercial value. Several species of Tenacibaculum have been identified as the causal agent of tenacibaculosis in fish, including Tenacibaculum maritimum, Tenacibaculum soleae, Tenacibaculum discolor, Tenacibaculum gallaicum, Tenacibaculum dicentrarchi and "Tenacibaculum finnmarkense" (quotations marks denote species that have not been validly published). Diagnosis of tenacibaculosis is usually based on culture-dependent detection and identification techniques which are time-consuming and do not allow to differentiate closely related species. The development of reliable techniques for studying the relationships between members of the genus Tenacibaculum and for distinguishing fish-pathogenic species of Tenacibaculum genus is, therefore, a key step in understanding the diversity and incidence of tenacibaculosis in global aquaculture, designing effective prevention strategies and early implementation of infection control measures. In this review, recent advances in molecular, serological, proteomic and chemotaxonomic techniques developed for the identification and differentiation of Tenacibaculum species, as well as for the analysis of their genetic and epidemiological relationships are discussed. Key features of current diagnostic methods likely to facilitate control and prevention of tenacibaculosis and to avoid the spread of its aetiological agents are also outlined.

Tenacibaculum todarodis sp. nov., isolated from a squid.

A Gram-stain-negative, rod-shaped, aerobic bacterial strain, designated LPB0136T, was isolated from a squid Todarodes pacificus, caught in the East Sea, off Korea. LPB0136T contained a circular chromosome of 3.02 Mb with a DNA G+C content of 30.7 mol%. The genome included 2726 protein-coding genes and three copies of rRNA operons. Phylogenetic analysis of the 16S rRNA gene sequence indicated that this isolate represents a member of the genus Tenacibaculum with an independent genomic species status, showing sequence similarities of 95.9 % to Tenacibaculum aestuarii SMK-4T and Tenacibaculum caenipelagi HJ-26MT. The detected respiratory quinone (MK-6) and major polar lipid (phosphatidylethanolamine) were similar to the chemotaxonomic profile of other species of the genus Tenacibaculum. The major cellular fatty acids profile (iso-C15 : 0, iso-C15 : 0 3-OH and iso-C15 : 0G) was also similar to those of members of genus Tenacibaculum, but the contents and amounts differed from those of closely related neighbours. Many biochemical and physiological characteristics also distinguished the isolate from other species within the genus Tenacibaculum. On the basis of the pholyphasic taxonomic data determined in this study, strain LPB0136T represents a novel species of the genus Tenacibaculum, for which the name Tenacibaculum todarodis sp. nov. is proposed. The type strain is LPB0136T (=KACC 18887T=JCM 31564T).

Comparison of serological and molecular typing methods for epidemiological investigation of Tenacibaculum species pathogenic for fish.

In the present study, the potential of serological methods, the repetitive extragenic palindromic PCR (REP-PCR) and the enterobacterial repetitive intergenic consensus sequence PCR (ERIC-PCR) for the typing of the species Tenacibaculum maritimum, Tenacibaculum soleae and Tenacibaculum discolor was evaluated. Moreover, molecular and proteomic techniques were used to assess variability among strains belonging to different serotypes, as well as isolated from different host species and geographical areas. Slide agglutination and dot-blot assays demonstrated the lack of immunological relationships among Tenacibaculum species analyzed. The serotype O1 was predominant within T. maritimum isolates regardless of the fish species or geographical area. Two serotypes were distinguished within T. soleae isolates and at least one within T. discolor strains. Species- and strain-specific profiles were obtained from the analysis of T. maritimum, T. soleae and T. discolor by REP-PCR and ERIC-PCR, demonstrating their potential as diagnostic tools. The genotyping analysis using both techniques showed genetic variability among the strains of each fish pathogenic Tenacibaculum species analysed. However, Tenacibaculum strains isolated from different host species or geographical areas or belonging to different serotypes produced REP and ERIC profiles with high similarity. Analysis by MALDI-TOF-MS of the T. maritimum strains could not detect any serotype-identifying biomarkers. Serotype-specific mass peaks were found for the serotypes O1 and O2 of T. soleae and for the serotype O1 of T. discolor. However, no relationships between the proteomic profiles and the source of isolation of the strains were obtained for any of the Tenacibaculum species analysed in this study.

First description of a mortality event in adult Pacific oysters in Italy associated with infection by a Tenacibaculum soleae strain.

Summer mortality episodes in adult Pacific oysters have been described since the 1950s in various farming areas. Starting in 2012, a recrudescence of mortalities in commercial-sized oysters was first observed in France and then in Italy, with seasonality extension and translation later in the year. Moribund individuals collected during an event in Italy in December 2014 showed yellowish lesions of the mantle and adductor muscle. Histological examination revealed filamentous bacteria associated with necrotic areas. Quantitative PCRs targeting OsHV-1 and Vibrio aestuarianus detected only high loads of the pathogenic bacteria in tissues of symptomatic individuals. A lower diversity of the hemolymph microbiota was also evidenced in moribund individuals, with a predominance of Vibrio and Arcobacter species. A strain of Flavobacteriaceae was isolated from all the symptomatic individuals. Sequence analysis of the 16S rRNA gene identified the strain as Tenacibaculum soleae. When strain pathogenicity was tested by injection in adult individuals, it induced mortality rates of up to 45%, even in the absence of V. aestuarianus. As mortality occurred only 11 days post-infection, further investigation is needed to determine its effective virulence in natural conditions. This is the first description of a Tenacibaculum strain associated with bivalve mortalities.

Tenacibaculum insulae sp. nov., isolated from a tidal flat.

A Gram-stain-negative, aerobic, non-motile, non-spore-forming bacterial strain, designated JDTF-31T, was isolated from a tidal flat in Jindo, a South Korean island. Strain JDTF-31T grew optimally at 25 °C and in the presence of 2.0 % (w/v) NaCl. The neighbour-joining phylogenetic tree based on 16S rRNA gene sequences revealed that strain JDTF-31T fell within the cluster comprising the type strains of Tenacibaculum species, joining the type strain of Tenacibaculum soleae. The novel strain exhibited 16S rRNA gene sequence similarity values of 98.3, 97.8 and 97.1 % to the type strains of T. soleae, Tenacibaculum haliotisand Tenacibaculum ovolyticum, respectively, and of 94.2-96.8 % to the type strains of the other Tenacibaculum species. Strain JDTF-31T contained MK-6 as the predominant menaquinone and iso-C15 : 0 and iso-C15 : 0 3-OH as the major fatty acids. The major polar lipids of strain JDTF-31T were phosphatidylethanolamine, one unidentified lipid and one unidentified aminophospholipid. The DNA G+C content of strain JDTF-31T was 31.3 mol% and its DNA-DNA relatedness values with the type strains of T. soleae, T. haliotis and T. ovolyticum were 16-27 %. The differential phenotypic properties, together with its phylogenetic and genetic data, revealed that strain JDTF-31T is separated from other recognized species of the genus Tenacibaculum. On the basis of the data presented, strain JDTF-31T represents a novel species of the genus Tenacibaculum, for which the name Tenacibaculuminsulae sp. nov. is proposed. The type strain is JDTF-31T (=KCTC 52749T=NBRC 112783T).

Tenacibaculum aestuariivivum sp. nov., isolated from a tidal flat.

A Gram-stain-negative, aerobic, non-motile, non-spore-forming bacterial strain, designated JDTF-79T, was isolated from a tidal flat in Jindo, an island of South Korea, and subjected to a taxonomic study using a polyphasic approach. Strain JDTF-79T grew optimally at 25 °C and in the presence of 2.0 % (w/v) NaCl. Phylogenetic trees based on 16S rRNA gene sequences showed that strain JDTF-79T fell within the clade comprising the type strains of species of the genus Tenacibaculum, clustering with the type strains of Tenacibaculum dicentrarchi, Tenacibaculum ovolyticum, 'Tenacibaculum haliotis' and Tenacibaculum soleae. The novel strain exhibited highest 16S rRNA gene sequence similarity (98.3 %) to the type strain of T. dicentrarchi and sequence similarities of 93.5-96.9 % to the type strains of the other species of the genus Tenacibaculum. Strain JDTF-79T contained MK-6 as the predominant menaquinone and anteiso-C15 : 0, iso-C15 : 0 3-OH and iso-C15 : 0 as the major fatty acids. The major polar lipids of strain JDTF-79T were phosphatidylethanolamine, one unidentified lipid and one unidentified aminophospholipid. The DNA G+C content of strain JDTF-79T was 30.3 mol%. Strain JDTF-79T had a mean DNA-DNA relatedness value of 19 % with the type strain of T. dicentrarchi. The differential phenotypic properties, together with the phylogenetic and genetic data, revealed that strain JDTF-79T is separated from other recognized species of the genus Tenacibaculum. On the basis of the data presented, strain JDTF-79T is considered to represent a novel species of the genus Tenacibaculum, for which the name Tenacibaculum aestuariivivum sp. nov. is proposed. The type strain is JDTF-79T (=KCTC 52980T=NBRC 112903T).

Tenacibaculum agarivorans sp. nov., an agar-degrading bacterium isolated from marine alga Porphyra yezoensis Ueda.

A novel Gram-stain-negative, aerobic, rod-shaped, non-flagellated and agar-digesting marine bacterium, designated as HZ1T, was isolated from the marine alga Porphyra yezoensis Ueda (AST58-103) collected from the coastal area of Weihai, PR China. Phylogenetic analysis based on 16S rRNA gene sequences placed HZ1T in the genus Tenacibaculum, and it formed a distinct clade in the phylogenetic tree with the type strains of Tenacibaculum amylolyticum and Tenacibaculum skagerrakense, with 97.0 % and 96.7 % 16S rRNA gene sequence similarities, respectively. The DNA G+C content of the isolate was 31.8 mol%. HZ1T contained MK-6 as the predominant menaquinone and iso-C15 : 0, summed feature 3 (C16 : 1ω7c and/or C16 : 1ω6c), iso-C17 : 0 3-OH and iso-C15 : 1G as the major fatty acids. The major polar lipids were phosphatidylethanolamine, four unidentified lipids and five unidentified aminolipids. On the basis of the results of the phylogenetic analysis and phenotypic properties, it is concluded that HZ1T represents a novel species of the genus Tenacibaculum, for which the name Tenacibaculumagarivorans sp. nov. is proposed. The type strain is HZ1T (=MCCC 1H00174T=KCTC 52476T).

Tenacibaculum haliotis sp. nov., isolated from the gut of an abalone Haliotis discus hannai.

A Gram-stain-negative, non-flagellated, gliding, non-spore-forming bacterial strain, designated RA3-2T, was isolated from the gut of an abalone (Haliotis discus hannai) collected from the sea around Jeju island, South Korea, and subjected to a polyphasic taxonomic study. RA3-2T grew optimally at 20 °C and in the presence of 2.0-3.0 % (w/v) NaCl. Phylogenetic trees based on 16S rRNA gene sequences indicated that RA3-2T fell within the clade comprising the type strains of species of the genus Tenacibaculum, clustering with the type strains of Tenacibaculum soleae, Tenacibaculum ovolyticum and Tenacibaculum dicentrarchi; showing 16S rRNA gene sequence similarity values of 96.2-96.8 %. The novel strain exhibited 16S rRNA gene sequence similarity values of 93.5-96.9 % to the type strains of the other species of the genus Tenacibaculum. RA3-2T contained MK-6 as the predominant menaquinone and iso-C15 : 0 and iso-C15 : 0 3-OH as the major fatty acids. The major polar lipids of RA3-2T were phosphatidylethanolamine, two unidentified lipids, one unidentified aminophospholipid and one unidentified glycolipid. The DNA G+C content of RA3-2T was 31.7 mol%. The differential phenotypic properties, together with the phylogenetic data, revealed that RA3-2T is separated from other species of the genus Tenacibaculum with validly published names. On the basis of the data presented, RA3-2T is considered to represent a novel species of the genus Tenacibaculum, for which the name Tenacibaculum haliotis sp. nov. is proposed. The type strain is RA3-2T (=KCTC 52419T=NBRC 112382T).

Primary Isolation and Characterization of Tenacibaculum maritimum from Chilean Atlantic Salmon Mortalities Associated with a Pseudochattonella spp. Algal Bloom.

This study presents the first isolation of Tenacibaculum maritimum from farmed Atlantic Salmon Salmo salar in Chile. The isolate, designated T. maritimum Ch-2402, was isolated from gills of Atlantic Salmon at a farm located in region X, Los Lagos, Chile, during the harmful algal bloom caused by Pseudochattonella spp. in February 2016. The algal bloom is reported to have caused 40,000 metric tons of mortality in this salmon farming area. The bacterium T. maritimum, which causes tenacibaculosis, is recognized as an important pathogen of marine fish worldwide. Genetic, phylogenetic, and phenotypic characterizations were used to describe the T. maritimum Ch-2402 isolate. The isolate was similar to the type strain of T. maritimum but was genetically unique. Tenacibaculum dicentrarchi isolates were also recovered during sampling from the same farm. Based on the fact that T. maritimum has been shown to cause disease in Atlantic Salmon in other regions, the presence of this bacterium poses a potential risk of disease to fish in the Chilean aquaculture industry. Received November 6, 2016; accepted May 29, 2017.

MALDI-TOF mass spectrometry for rapid differentiation of Tenacibaculum species pathogenic for fish.

Tenacibaculosis is a fish disease that limits the culture of a variety of marine fish species of commercial value in the world. The genus Tenacibaculum includes several species, and their discrimination is of clinical interest in order to improve the management of an outbreak of the disease. In this study, a novel proteomic approach based on matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) analysis was evaluated for the identification and differentiation of Tenacibaculum species. The peak mass lists derived from MALDI-TOF-MS analysis were examined for the detection of potential biomarkers, similarity and cluster analysis and principal component analysis (PCA). Culture media used for bacterial growth did not affect the mass fingerprints. Eight genus-specific peaks were found in all the Tenacibaculum species analysed. Moreover, at least one species-specific peak was found in the species Tenacibaculum maritimum, Tenacibaculum soleae, Tenacibaculum dicentrarchi, Tenacibaculum litoreum and Tenacibaculum ovolyticum. These peaks could serve as biomarkers for the rapid identification of these bacterial fish pathogens. The cluster and PCA clearly separated the species T. maritimum, T. soleae, T. dicentrarchi and T. ovolyticum in different clusters. However, species of Tenacibaculum discolor and Tenacibaculum gallaicum were difficult to distinguish based on their protein fingerprints. To our knowledge, this is the first study that deals with the characterization and determination of biomarkers of Tenacibaculum species by MALDI-TOF mass spectrometry. This approach proved to be an effective and reliable technique for the discrimination of the Tenacibaculum species; therefore, it could be integrated as a routine diagnostic tool in microbiological laboratories.