Characterizing skin mucus barrier properties of Atlantic salmon by the use of multiple particle tracking

Due to the increasing demand of fish as a source of protein worldwide, commercial aquaculture is rapidly expanding. Farmed fish are however exposed to stressful situations, and bacterial infections represent a significant problem. As antimicrobial treatments are not considered a sustainable alternative of treatment, it is imperative to develop new preventative practices. The mucus layer that cover fish integument represents the first line of defence against pathogen invasion, and knowledge of its barrier properties is considered to be of great importance in the attempt to develop such preventative practices. This thesis investigates the barricading properties of germ free and conventional raised Atlantic salmon fry through multiple particle tracking of particles and bacteria in ex vivo and in vitro mucus models.
To examine mucus adhesive and viscoelastic properties at bacterial length scales the barrier properties of germ free and conventional raised fish skin mucus to particle transport were investigated. The significance of surface chemistry on particle diffusion was explored by using negatively charged carboxylated nanoparticles and PEGylated nanoparticles of the same size (1µm). Particle transport was found to be significantly dependent on surface charge, with PEGylated particles showing less restricted mobility on both germ free and conventional raised fish skin mucus. Further, the PEGylated particles did not exhibit mobility-patterns corresponding to that of Brownian motion, indicating that bacterial mobility was likely to be restricted by both charge interactions and physical obstruction by the mucus mesh.
Germ free and conventional raised fish were incubated for 24 hours and 72 hours to examine bacterial movements and determine how prolonged incubation time affected mucosal barrier properties. Bacterial mobility was found to be severely restricted in both germ free and conventional raised fish after 24 hours of incubation compared to their mobility in tryptic soy broth, reflecting mucosal barricading properties to bacteria. Further, the bacteria were found to have increased mobility on germ free and conventional raised fish after 72 hours of incubation, indicating a reduction in mucus viscosity. Z-stack analysis further showed an increased mean distance between the bacteria and the fish skin, suggesting an increase in mucus volume. Bacteria on germ free fish was found to have less restricted mobility at both incubation time-points, compared to the conventional raised fish, indicating that mucus commensal bacteria influence mucus barrier properties.
Bacteria and particles were also tracked in vitro in collected mucus secretions from conventional raised and germ free fish, to examine how it resembled mucus properties found through the ex vivo tracking. Particle tracking revealed that the collected mucus samples were generally of a less viscous character than in the ex vivo tracking`s. The in vitro tracking in collected mucus from germ free fish was found to be more similar with the ex vivo tracking on germ free fish skin mucus, compared to the conventional raised in vitro and ex vivo tracking’s. In vitro bacterial tracking’s revealed the same trend. Tracking`s conducted on collected mucus from germ free fish was found to have similar barricading properties as the ex vivo tracking`s on germ free fish incubated for 72 hours indicated. These observations further confirmed the reduced viscosity of germ fry mucus, and are indicative of reduced barrier properties in the germ free fish.