Towards sustainable eel farming: Immunostimulation strategies against the zoonotic pathogen vibrio vulnificus
- Autores: Carla Molina Sorribes
- Directores de la Tesis: Eva Sanjuán Caro (dir. tes.), Belén Fouz Rodriguez (codir. tes.), Carmen Amaro González (codir. tes.)
- Lectura: En la Universitat de València ( España ) en 2025
- Idioma: inglés
- Tribunal Calificador de la Tesis: Sergi Ferrer Soler (presid.), Carla Hernández Cabañero (secret.), Ana María Silva do Vale (voc.)
- Programa de doctorado: Programa de Doctorado en Biomedicina y Biotecnología por la Universitat de València (Estudi General)
- Materias:
- Enlaces
- Tesis en acceso abierto en: TESEO
- Resumen
Stress experience by animals in aquaculture facilities increases their susceptibility to infectious diseases, leading to the frequent use of antibiotics to control outbreaks. Among the most concerning pathogens is Vibrio vulnificus, an aquatic zoonotic bacterium whose geographical distribution is expanding due to global warming. This microorganism causes what is known as warm-water vibriosis in fish, with its most severe manifestation being hemorrhagic septicemia, which has high mortality rates, especially in eels (Anguilla anguilla) - its primary host. The species is divided into five lineages with pathogenic potential for humans and a polyphyletic pathovar (pv. piscis), specifically adapted to infected fish. Its virulence in fish is associated with the pVir plasmid, which contains genes such as fpcrp and ftbp. These genes form an essential system to evade the innate immunity of fish and act as a "blood survival kit." Moreover, the conditions in fish farms have influenced the evolution of this pathogen, as two of its five lineages emerged in these facilities as human-pathogenic variants following successive vibriosis outbreaks. For these reasons, developing sustainable strategies to control infections is a priority. These strategies should also act as an evolutionary bottleneck against the horizontal transfer of adaptation genes by limiting the emergence of more resilient strains and; thus, contributing to improving human, animal, and environmental health. This doctoral thesis focused on developing two main preventive strategies in aquaculture - functional feeding and vaccination - whose specific aim is to control vibriosis outbreaks caused by V. vulnificus. First, the bactericidal effect of various microencapsulated essential oil blends was evaluated against different V. vulnificus pv. piscis strains belonging to distinct lineages. After selecting the two most effective formulations, they were incorporated into the diet of eels to assess their immunostimulant potential. The results demonstrated that the diet supplemented with the UV-1 phytobiotic significantly increased serum lysozyme activity after five weeks of administration. Subsequently, its protective effect against vibriosis was evaluated, showing an improved eel survival rate at both assessed concentrations, provided that the animals had been previously acclimated to the control diet for at least four weeks. Moreover, the fact of this phytobiotic also exhibited protective effect against Vibrio parahaemolyticus in shrimp, supports its potential as a broad-spectrum strategy for controlling bacterial diseases in aquaculture-relevant species. Concurrently, a vaccine strategy based on the common proteins Fpcrp and Ftbp proteins, which have been identified in different pathogenic species, including V. vulnificus, V. harveyi, and Photobacterium damselae subsp. damselae., was developed. After selecting and optimizing the sequences of the fpcrp and ftbp genes, they were cloned into two different expression vectors, establishing optimal conditions for producing recombinant proteins. The purification of these recombinant proteins allowed as to evaluate eel response to them. Its combination demonstrated immunogenic potential, inducing a significant increase in lysozyme activity and the production of specific antibodies. However, only intraperitoneal immunization with each antigen separately showed the capacity to improve survival. Once the immunogenic and protective effects of these antigens were confirmed, the use of inclusion bodies as delivery platforms was explored, as these protein structures have proven suitable for antigen incorporation into diets due to their resistance to high temperatures and acidic pH. Subsequent vaccination trials via oral intubation and immersion revealed that both are suitable routes to induce an increase in lysozyme activity, plasma antibody production, and moderate survival improvement. Nonetheless, the observed protection patterns differed depending on the administration route: while the formulation combining both antigens was more effective when administered via oral intubation, the vaccine based on rFtbp was more effective when applied by immersion. Although the results obtained are promising, further studies are required to determine whether increasing the antigen dose could enhance the protective efficacy of the develop formulations. Additionally, it is necessary to evaluate the broad-spectrum potential of this vaccination approach in other fish species and against other bacteria, considering that the selected antigens are present in other bacterial species and play a key role in fish bloodstream survival. All in all, the findings of this work contribute to the development of sustainable tools for disease control in aquaculture, offering alternatives to antibiotic use that could have a positive impact on global health.
