Oral vaccination of fish against vibriosis using spore-display technology.

Oral vaccines are highly demanded by the aquaculture sector, to allow mass delivery of antigens without using the expensive and labor-intensive injectable vaccines. These later require individual handling of fish, provoking stress-related mortalities. One possible strategy to create injection-free vaccine delivery vehicles is the use of bacterial spores, extremely resistant structures with wide biotechnological applications, including as probiotics, display systems, or adjuvants. Bacterial spores, in particular those of Bacillus subtilis, have been shown to behave as mucosal vaccine adjuvants in mice models. However, such technology has not been extensively explored against fish bacterial disease. In this study, we used a laboratory strain of B. subtilis, for which a variety of genetic manipulation tools are available, to display at its spores surface either a Vibrio antigenic protein, OmpK, or the green fluorescence protein, GFP. When previously vaccinated by immersion with the OmpK- carrying spores, zebrafish survival upon a bacterial challenge with V. anguillarum and V. parahaemolyticus, increased up to 50 - 90% depending on the pathogen targeted. Further, we were able to detect anti-GFP-antibodies in the serum of European seabass juveniles fed diets containing the GFP-carrying spores and anti-V. anguillarum antibodies in the serum of European seabass juveniles fed the OmpK-carrying spores containing diet. More important, seabass survival was increased from 60 to 86% when previously orally vaccinated with in-feed OmpK- carrying spores. Our results indicate that B. subtilis spores can effectively be used as antigen-carriers for oral vaccine delivery in fish.

Lactobacillus plantarum in Dual-Species Biofilms With Listeria monocytogenes Enhanced the Anti-Listeria Activity of a Commercial Disinfectant Based on Hydrogen Peroxide and Peracetic Acid.

The aim of this work was to investigate the effect of dual-species biofilms of Listeria monocytogenes with Lactobacillus plantarum on the anti-Listeria activity of a hydrogen peroxide/peracetic acid based commercial disinfectant (P3, Oxonia) when using conditions approaching the food industry environment. Nine strains of L. monocytogenes, including eight persistent strains collected from the meat industry and one laboratory control strain, were used in mono and in dual-species biofilms with a strain of L. plantarum. Biofilms were produced on stainless steel coupons (SSCs), at 11°C (low temperature) or at 25°C (control temperature), in TSB-YE (control rich medium) or in 1/10 diluted TSB-YE (mimicking the situation of biofilm formation after a deficient industrial cleaning procedure). The biofilm forming ability of the strains was evaluated by enumeration of viable cells, and the antibiofilm activity of P3 was assessed by the log reduction of viable cells on SSC. In both nutrient conditions and at low temperature, there was no significant difference (p > 0.05) between L. monocytogenes biofilm forming ability in mono- and in dual-species biofilms. In dual-species biofilms, L. monocytogenes was the dominant species. However, it was generally more susceptible to the lower concentration of P3 0.5% (v/v) than in pure culture biofilms. The presence of L. plantarum, although without significant interference in the number of viable cells of L. monocytogenes, enhanced the efficacy of the anti-Listeria activity of P3, since dual-species biofilms were easier to control. The results presented here reinforce the importance of the investigation into co-culture biofilms produced in food industry conditions, namely at low temperatures, when susceptibility to sanitizers is being assessed.