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.publishedVersio

Experimental induction of mouthrot in Atlantic salmon smolts using Tenacibaculum maritimum from Western Canada

Mouthrot, or bacterial stomatitis, is a disease which mainly affects farmed Atlantic salmon, (Salmo salar, L.), smolts recently transferred into salt water in both British Columbia (BC), Canada, and Washington State, USA. It is a significant fish welfare issue which results in economic losses due to mortality and antibiotic treatments. The associated pathogen is Tenacibaculum maritimum, a bacterium which causes significant losses in many species of farmed fish worldwide. This bacterium has not been proven to be the causative agent of mouthrot in BC despite being isolated from affected Atlantic salmon. In this study, challenge experiments were performed to determine whether mouthrot could be induced with T. maritimum isolates collected from outbreaks in Western Canada and to attempt to develop a bath challenge model. A secondary objective was to use this model to test inactivated whole‐cell vaccines for T. maritimum in Atlantic salmon smolts. This study shows that T. maritimum is the causative agent of mouthrot and that the bacteria can readily transfer horizontally within the population. Although the whole‐cell oil‐adjuvanted vaccines produced an antibody response that was partially cross‐reactive with several of the T. maritimum isolates, the vaccines did not protect the fish under the study's conditions.publishedVersio

Tenacibaculosis in Norwegian farmed Atlantic salmon

Tenacibaculosis is a bacterial ulcerative skin disease of many economically important farmed fish species worldwide caused by members of genus Tenacibaculum. For the Norwegian salmon farming industry, however, tenacibaculosis was not recognized as an important disease until 2010. Since then, the increased use of media supporting Tenacibaculum growth has resulted in the identification of Tenacibaculum spp. from a large number of outbreaks of skin lesions/ulcers. Bacterial skin infections that cause skin lesions/ulcers in farmed Atlantic salmon are not only important fish health and welfare issues that needs to be solved, but also cause significant economic losses. From investigations of tenacibaculosis outbreaks in the northernmost parts of Norway, a Tenacibaculum sp. strain was repeatedly identified over several years at multiple farms. Sequence similarity analysis showed that this Tenacibaculum sp. strain was most closely related to Tenacibaculum dicentrarchi, but different enough to constitute a novel Tenacibaculum species. A polyphasic investigation showed that this was indeed a novel species in the genus Tenacibaculum, for which the name Tenacibaculum finnmarkense was proposed. The delineation of T. finnmarkense and T. dicentrarchi has since been further demonstrated by whole genome analysis. In September 2015, two separate outbreaks of tenacibaculosis occurred at two Atlantic salmon farms in Northern Norway. The events resulted in major losses of smolts newly transferred into seawater. Prior to, and during the outbreaks, large numbers of small jellyfish, identified as Dipleurosoma typicum (Boeck) were observed in the vicinity of the farms and inside the net-pens. During these outbreaks, T. finnmarkense was found to be the dominant bacteria associated with the outbreaks. Moreover it was shown that D. typicum were unlikely to be a vector for T. finnmarkense. However, it was shown that the jellyfish caused direct damage to the fish’s skin which likely exacerbated the bacterial infection by allowing an entry point for T. finnmarkense. Bath challenges conducted during this study using T. finnmarkense and Atlantic salmon smolts showed that for the first time this bacterium was able to induce tenacibaculosis as it presents in the field without pre-stressors or co-infection. The study also showed that there is pathogenic variation between T. finnmarkense strains, with T. finnmarkense strain HFJT being the most pathogenic. A cohabitation experiment showed that T. finnmarkense does not appear to spread horizontally from fish to fish and more work is therefore required to determine possible reservoirs and/or vectors for this bacterium. Due to the success of vaccines targeting other bacterial pathogens in Norwegian farmed Atlantic salmon, the efficacy of a whole cell inactivated vaccine targeting T. finnmarkense was tested. Despite the vaccines inducing an antibody response, they did not manage to give any protection against tenacibaculosis induced through a bath infection. Future research needs to focus on mitigation tools, which will need to include refining the challenge model necessary for testing such tools

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

First isolation, identification and characterisation of Tenacibaculum maritimum in Norway, isolated from diseased farmed sea lice cleaner fish Cyclopterus lumpus L

The use of cleaner fish as biological controls of salmon lice (Lepeophtheirus salmonis) has increased exponentially in the last decade in Norwegian Atlantic salmon (Salmo salar) production. This alternative to chemical treatments has resulted in the emergence of lumpsucker (Cyclopterus lumpus) hatcheries and culture facilities in Norway. It has been shown that the use of lumpsuckers can be an effective, biological approach for the removal of salmon lice, but it has also been shown that there are a number of biological challenges (i.e. parasites and bacteria) with the production and use of these fish. This study describes the first case of isolation of Tenacibaculum maritimum, a significant fish pathogen worldwide, in cultured juvenile lumpsuckers in Norway. The fish were lethargic and showed skin lesions characterised by increased mucus production and presence of whitish necrotic tissue especially in the head region. Skin scrapings revealed large amounts of bacteria dominated by rod-shaped Tenacibaculum-like bacteria, which were shown to be closely related to T. maritimum type strain through genetic and phenotypic characterisation. Histopathological analysis showed that the bacteria was closely associated with the pathology and therefore could be contributing to the disease and/or mortality

Genotyping of Tenacibaculum maritimum isolates from farmed Atlantic salmon in Western Canada

Mouthrot infections (bacterial stomatitis) have a significant impact on the Atlantic salmon aquaculture industry in Western Canada due to economic losses and fish welfare. Bacteria isolated from lesions in the field have been identified as Tenacibaculum maritimum. Mouthrot is different to classical tenacibaculosis, which is most commonly associated with ulcerative lesions, frayed fins and tail rot. The marine fish pathogen T. maritimum is found worldwide; however, in Western Canada, the knowledge of the genetic profile of T. maritimum is limited. This study looked at increasing this knowledge by genotyping T. maritimum isolates collected from Atlantic salmon from farms in Western Canada. These genotypes were compared to other species of the genus Tenacibaculum, as well as other known sequence types within the species. The Western Canadian isolates belong to two new sequence types within the T. maritimum species. Phylogenetic analysis shows that the isolates form a distinct branch together with T. maritimum NCIMB 2154T separate from other Tenacibaculum type strains, and they are most closely related to strains from Norway and Chile

Concurrent jellyfish blooms and tenacibaculosis outbreaks in Northern Norwegian Atlantic salmon (Salmo salar) farms

Tenacibaculosis is an increasing problem in the Norwegian Atlantic salmon aquaculture industry causing significant economic losses. In September 2015, two separate outbreaks of suspected tenacibaculosis occurred at two Atlantic salmon farms in Finnmark County in Northern Norway. The events resulted in major losses of smolts newly transferred into seawater. Prior to, and during the outbreaks, large numbers of small jellyfish, identified as Dipleurosoma typicum (Boeck) were observed in the vicinity of the farms and inside the net-pens. This study investigates the possible link between the jellyfish, Tenacibaculum spp. and the tenacibaculosis outbreaks. Bacteriology, histology, scanning and transmission electron microscopy, and real-time RT-PCR screening were performed on both fish and jellyfish samples. Based on the findings, Tenacibaculum finnmarkense was found to be the dominant bacteria associated with the tenacibaculosis outbreaks at both sites and that D. typicum is unlikely to be a vector for this fish pathogenic bacterium. However, results do show that the jellyfish caused direct damage to the fish’s skin and may have exacerbated the bacterial infection by allowing an entry point for bacteria

Tenacibaculum finnmarkense sp. nov., a fish pathogenic bacterium of the family Flavobacteriaceae isolated from Atlantic salmon

A novel Gram-stain negative, aerobic, non-flagellated, rod-shaped gliding bacterial strain, designated HFJT, was isolated from a skin lesion of a diseased Atlantic salmon (Salmo salar L.) in Finnmark, Norway. Colonies were observed to be yellow pigmented with entire and/or undulating margins and did not adhere to the agar. The 16S rRNA gene sequence showed that the strain belongs to the genus Tenacibaculum (family Flavobacteriaceae, phylum ‘Bacteroidetes’). Strain HFJT exhibits high 16S rRNA gene sequence similarity values to Tenacibaculum dicentrarchi NCIMB 14598T (97.2 %). The strain was found to grow at 2–20 °C and only in the presence of sea salts. The respiratory quinone was identified as menaquinone 6 and the major fatty acids were identified as summed feature 3 (comprising C16:1 ω7c and/or iso-C15:0 2-OH), iso-C15:0, anteiso-C15:0, iso-C15:1 and iso-C15:0 3-OH. The DNA G+C content was determined to be 34.1 mol%. DNA–DNA hybridization and comparative phenotypic and genetic tests were performed with the phylogenetically closely related type strains, T. dicentrarchi NCIMB 14598T and Tenacibaculumovolyticum NCIMB 13127T. These data, as well as phylogenetic analyses, suggest that strain HFJT should be classified as a representative of a novel species in the genus Tenacibaculum, for which the name Tenacibaculum finnmarkense sp. nov. is proposed; the type strain is HFJ T = (DSM 28541T = NCIMB 42386T)

Pathology of experimentally induced mouthrot caused by Tenacibaculum maritimum in Atlantic salmon smolts

Mouthrot, caused by Tenacibaculum maritimum is a significant disease of farmed Atlantic salmon, Salmo salar on the West Coast of North America. Smolts recently transferred into saltwater are the most susceptible and affected fish die with little internal or external clinical signs other than the characteristic small (usually < 5 mm) yellow plaques that are present inside the mouth. The mechanism by which these smolts die is unknown. This study investigated the microscopic pathology (histology and scanning electron microscopy) of bath infected smolts with Western Canadian T. maritimum isolates TmarCan15-1, TmarCan16-1 and TmarCan16-5 and compared the findings to what is seen in a natural outbreak of mouthrot. A real-time RT-PCR assay based on the outer membrane protein A specific for T. maritimum was designed and used to investigate the tissue tropism of the bacteria. The results from this showed that T. maritimum is detectable internally by real-time RT-PCR. This combined with the fact that the bacteria can be isolated from the kidney suggests that T. maritimum becomes systemic. The pathology in the infected smolts is primarily mouth lesions, including damaged tissues surrounding the teeth; the disease is similar to periodontal disease in mammals. The pathological changes are focal, severe, and occur very rapidly with little associated inflammation. Skin lesions are more common in experimentally infected smolts than in natural outbreaks, but this could be an artefact of the challenge dose, handling and tank used during the experiments