Investigation into the Cytotoxicity of Soluble Extracellular Products Secreted by Tenacibaculum maritimum and other Tenacibaculum species
Creators: Michnik, Matthew LC
Publisher: name: University of Alberta Library
Year: 2024

<p>Atlantic salmon (Salmo salar) aquaculture in British Columbia (BC), Canada is the province’s largest agricultural export and is a significant contributor to its economy. As with any food production industry, aquaculture faces challenges regarding disease. One disease of interest is tenacibaculosis, locally known as mouth rot or yellow mouth. Antibiotics are the only treatment for this infection, which results in mouth rot being responsible for the majority of antibiotic use in BC aquaculture. Further, there is no available vaccine for preventing infections of Atlantic salmon and the pathogenesis mechanism of the disease is unknown. Current evidence supports Tenacibaculum maritimum as an opportunistic pathogen capable of causing mouth rot disease. However, it is unclear as to how this bacterium may establish infection, when it is most likely to cause an outbreak, and the mechanisms by which other related bacterial species could be involved? These questions won’t be answered in their entirety, but my goal is to advance our functional understanding of the associated disease bacteria by probing these overarching questions.<br>
    Previous studies have demonstrated that extracellular products (ECP) from T. maritimum are capable of causing fish muscle damage and cell death, implicating that these secreted products could be involved in disease pathogenesis. However, the mechanism by which these ECP work is not fully known. Our study uses a validated flow cytometry cell death quantification assay to determine the mechanism of salmonid cellular death after exposure to T. maritimum ECP. Cell lines originating from salmonid mucosal epithelial tissue were used as relevant models in cytotoxicity trials. When treated with T. maritimum ECP, these cells underwent programmed host cell death (apoptosis) due to the exposure. The impact of environmental conditions, such as salinity and temperature, on bacterial growth and the production of cytotoxic ECP were also examined. When investigated in vitro, both conditions appear to be involved in cytotoxicity, aligning with recent literature and industry field reports. The bacterial ECP underwent inactivation treatments of heat denaturation, protease inhibitors, and a metalloprotease inhibitor to narrow down the responsible product. Overall, this work pushes forward the knowledge on T. maritimum secreted ECP; however, there is still much to follow up with regarding these products and their involvement in field infections.<br>
    Mouth rot infections occur in seawater and on the skin of fish where there are plenty of other bacteria that may contribute to disease pathogenesis. Related Tenacibaculum species are known to be capable of causing tenacibaculosis infections in Atlantic salmon. I hypothesize that the contributions from different bacteria at the site of infection result in the disease symptomatology differences seen internationally. Herin, two mouth rot isolated Tenacibaculum species, Tenacibaculum dicentrarchi (known pathogen) and Tenacibaculum ovolyticum (unknown if pathogenic), were compared to T. maritimum regarding their various potentially pathogenic characteristics, such as biofilm production and ECP cytotoxicity. In this study, T. maritimum was the only isolate that demonstrated an ability to form biofilms. Surprisingly, when cells from a salmonid immune cell line were treated with soluble ECP from the various bacteria, T. dicentrarchi and T. ovolyticum had the highest cytotoxicity. Further, after the various ECP underwent inactivation trials (heat-treatment, protease inhibitors, and metalloprotease inhibitors), there were varied results between the potential pathogens, thereby suggesting the production of distinct virulence factors between the Tenacibaculum species. <br>
    Overall, this study contributes to the functional understanding of cytotoxic extracellular products secreted by T. maritimum and other Tenacibaculum species. While the degree of involvement of these secreted products remains unknown, the mechanism of their cytotoxicity on a cellular level is now uncovered. Further, the potential for cytotoxic contributions from related species was demonstrated in the current study and adds to the complexity of this disease. </p>