Jeremy Carson’s scientific contributions

The bacterial skin disease tenacibaculosis, caused by Tenacibaculum species, affects numerous economically important marine fish, including salmonids. This study reports the ability of three Tenacibaculum maritimum strains, belonging to different molecular O‐AGC types, and a single Tenacibaculum dicentrarchi strain to induce tenacibaculosis in farmed Chinook salmon ( Oncorhynchus tshawytscha , Walbaum 1792) in Aotearoa New Zealand. Naïve Chinook salmon were exposed to T. maritimum (2 × 10 ⁸ cells/mL) and T. dicentrarchi (2 × 10 ⁷ cells/mL) by immersion using natural seawater. Clinical signs of tenacibaculosis were apparent in all T. maritimum strains used in the challenged fish. Of these, 100% of the fish challenged with O‐AGC Type 2‐1 and Type 3‐2 strains became moribund, whereas only 60% of the O‐AGC Type 3‐0 challenged fish became moribund. Fish exposed to T. dicentrarchi showed more severe symptoms, exposing musculature in 51% of the challenged population, with 28% of fish becoming moribund. Gross pathological signs of fin rot, scale loss, skin ulcers and haemorrhagic skin spots were observed for both Tenacibaculum species and were consistent with those observed on farmed fish. Pure T. maritimum and T. dicentrarchi cultures were reisolated from epidermal damage of challenged fish. Tenacibaculum species was not isolated from the anterior kidney of affected fish, which indicates no systemic infection in Chinook salmon.

The bacterial skin disease tenacibaculosis, caused by Tenacibaculum species, can compromise numerous species of economically important marine fish, including salmonids. While tenacibaculosis is a known threat to Atlantic salmon ( Salmo salar ) aquaculture, the pathogenesis of Tenacibaculum maritimum and Tenacibaculum dicentrarchi on Chinook salmon ( Oncorhynchus tshawytscha ) has not yet been investigated. In this study, three molecular O-AGC types of T. maritimum (O-AGC Type 3-0, Type 2-1 and Type 3-2) and T. dicentrarchi isolated during a disease outbreak of farmed Chinook salmon in Aotearoa New Zealand were assessed for their ability to induce tenacibaculosis in salmon smolts under controlled conditions. Naive Chinook salmon were exposed to T. maritimum or T. dicentrarchi by immersion. Clinical signs of tenacibaculosis were apparent post-exposure and observed in 100% of all three molecular O-AGC types of T.-maritimum -challenged fish, with 100% morbidity in O-AGC Type 2-1 and Type 3-2 and 60% in O-AGC Type 3-0. Chinook salmon exposed to T. dicentrarchi showed characteristic clinical signs of disease in 51% of the challenged population, with 28% morbidity. Common gross pathological signs observed for both Tenacibaculum species were congruent with observations on farmed fish in the field, including scale loss, erythematous skin lesion, skin ulcers, fin necrosis, mouth erosion and gill ulceration. Exophthalmia was observed only in T. maritimum -challenged fish, while skin ulcers appeared grossly more severe with exposed musculature in T. dicentrarchi -challenged fish. Pure T. maritimum and T. dicentrarchi cultures were reisolated from the skin and gills of the challenged fish and their identity was confirmed by species-specific PCR and molecular O-AGC typing. Challenge experiments and associated field surveillance (for T. maritimum ) did not show the presence of culturable T. maritimum cells in the anterior kidney. This provides compelling evidence that tenacibaculosis in farmed Chinook salmon is an external infectious disease, and that Tenacibaculum is a marine obligate organism that is unable to survive in fish body fluids and does not cause septicaemia. This has repercussions for approaches to experimental challenges with Tenacibaculum species, which must occur by immersion rather than intraperitoneal or intramuscular inoculation, to replicate the natural transmission pathway and to ensure a successful challenge model. This study fulfilled modernised Koch’s postulates for the three molecular O-AGC types of T. maritimum and single strain of T. dicentrarchi as aetiological agents of tenacibaculosis in Chinook salmon that cause mortalities with considerable external abnormalities. Author summary Chinook salmon, Oncorhynchus tshawytscha , is the most significant species of Pacific salmon for its large size and nutritional content which makes it a premium choice for aquaculture. In Aotearoa|New Zealand, Chinook salmon is the only marine salmon species farmed. For a decade, the industry was impacted by an undiagnosed skin disease resulting in high mortalities. Disease susceptibility in Chinook salmon is scarcely studied and added to the challenge for a timely diagnosis. This novel research provides insight on disease susceptibility of Chinook salmon and confirms Tenacibaculum species identified in New Zealand pose a high threat to the aquaculture industry. This research has global implications and contributes valuable insights and approaches to disease management that can be applied in British Columbia and Canada where Chinook salmon populations are in decline.

Tenacibaculum maritimum is a cosmopolitan bacterial pathogen with the potential to cause significant losses in a broad range of farmed and wild marine fish species. This study investigated the antigenic diversity of T. maritimum isolated in culture from farmed Chinook (king) salmon (Oncorhynchus tshawytscha) from the Marlborough Sounds in New Zealand. A total of 36 isolates were examined using antibody serotyping and rapid molecular serotyping via multiplex PCR (mPCR) targeting genes encoding O-antigen biosynthesis enzymes. Serological analysis using three different polyclonal antisera developed against T. maritimum isolated from farmed Atlantic salmon (Salmo salar) in Tasmania, Australia revealed that there are three putative serotypes of T. maritimum that occur in New Zealand. The predominant serotype was defined by a positive reaction to all three Tasmanian antisera (antisera A, B and C), designated as serotype ABC. This serotype was found at all nine farm locations tested and represented 81% of all isolates examined. The same library of isolates was evaluated by mPCR serotyping and found three O-AGC types among tested isolates. O-AGC Type 3 was not only the predominant type (72%) present, but it also had a wide distribution, having been isolated at eight of the nine farms. Two other O-AGC types (O-AGC Type 2–1 and O-AGC Type 3–2) were identified, providing evidence of genetic variation. However, there was only partial concordance between the two serotyping techniques, which is likely linked to differences in the way serotypes are defined in the two approaches that were used. Nevertheless, in broad terms there is good evidence of intraspecific antigenic variation within our library of isolates, and collectively these data will be of crucial importance for assessing the pathogenicity of the isolates and the subsequent development of a vaccine for this emerging disease in New Zealand marine salmon farms.

We evaluated the suitability of three diagnostic techniques to detect Tenacibaculum maritimum in farmed Chinook salmon (or ‘king’ salmon), Oncorhynchus tshawytscha, exhibiting clinical signs of tenacibaculosis with skin spots and ulcers. A selective bacterial culture technique using Marine Shieh's Selective Medium (MSSM) found 100% detection of T. maritimum in sampled farmed O. tshawytscha. In comparison, pathogen detection using a non-selective culture media (marine agar) revealed 33.3% detection of T. maritimum, while a droplet digital polymerase chain reaction (ddPCR) specific assay provided 81.5% positive detection. Skin abnormality type was a significant predictor of ddPCR detection, with spots less likely to be found positive compared to ulcers (p = 0.01). This study also reports the first isolation of co-occurring Tenacibaculum soleae and Tenacibaculum dicentrarchi from skin abnormalities of farmed O. tshawytscha in New Zealand. The 16S ribosomal RNA gene sequence amplified from representative isolates shared 100% and 99% and identity with T. soleae and T. dicentrarchi 16S rRNA sequences from GenBank, respectively. T. soleae and T. dicentrarchi colonies were morphologically distinct from T. maritimum. The MSSM culture technique facilitated rapid recovery of all three Tenacibaculum species within 48 h when incubated at room temperature (20–23 °C). In this study we discuss the advantages of a selective medium for surveillance of pathogenic Tenacibaculum species that infect fish when used in combination with confirmatory identification by molecular techniques, in contrast to relying only on PCR based detection methods. We suggest that culture dependent diagnostic techniques provide sufficiently sensitive, rapid, and cost-effective on-farm screening as this method detects only viable organisms which pose immediate risk to fish. The rapid identification and management of emerging fish-pathogenic species of Tenacibaculum in New Zealand is essential for the diagnosis of tenacibaculosis outbreaks which can impact farm production and animal welfare.