Przybylska, Dominika Alicja

For at sikre en sund og rentabel i akvakultur produktion er kontrol af fiskesygdomme af stor betydning. Muligheden for brug af medicin til behandling af almindelige infektiøse sygdomme bliver mere og mere begrænset og kostbare, og er i nogle tilfælde ikke mulig på grund af resistens i bakterier og svampe. Derfor er der stor fokus på alternativer til medicinering, som kan reducere risikoen for sygdomme og forbedre fiskevelfærden gennem stimulering af det ikke-specifikke forsvarssystem. ß-glucaner er naturligt forekommende polysaccharider, der findes i cellevæggen hos planter, bakterier og svampe. ß-glucaner er nogle af de mest effektive og bedst undersøgte stoffer med immunmodulerende egenskaber i fisk. ß-glucaner har vist sig at forbedre immunsystemet og farmakologisk klassificeres de som biologiske respons-modifikatiorer. Fokus i denne afhandling er: 1. Udvikling af en model til undersøgelse af den biologiske virkning af to kommercielt tilgængelige ß-glucan berigede produkter på sårhelingsprocessen i almindelig karpe (Cyprinus carpio L.) under sterile, kontrollerede betingelser; 2. Undersøgelse af betydningen af intravenøst injiceret ß-glucan på det mukosale-immunrespons og en antistof switch-lignende proces i karper. For at nå disse mål, blev der anvendt forskellige metoder, såsom kvantitativ realtime-PCR (RT-PCR) til måling af ekspressionen af immun-relaterede gener involveret i sårhelingsprocessen, ELISA for bestemmelse af specifikke antistoftitere samt cortisol-måling af stressniveau, real-time respiratorisk burst assay til bestemmelse af produktionen af frie-radikaler og billedanalyse af sårhelingsprocessen. Det er blevet vist at behandlede sygdomsudbrud giver anledning til ændringer i kvalitetsparametre som tekstur i frisk fiskekød. Hvilket har været udgangspunktet for yderligere at undersøge anvendelses af immunmodulatoren ß-glucaner. Den videre undersøgelse af effekter af ß-glucan har vist, at to kommercielt tilgængelige ß-glucan berigede produkter, MacroGard og 6,3 kDa havrefibre, har en direkte positiv effekt på sårlukning i karper ved badbehandling med disse. Således var sårheling hurtigere i ß-glucan behandlede fisk i sammenligning med ikke-behandlede fisk. Det immunologiske og regenerative respons efter stimulering med PAMPs (Pathogenassociated molecular patterns) og DAMPs (danger-associated molecular patterns molecules) under kontrollerede forhold, uden at eksponeringen for patogener er ligeledes blevet undersøgt, og en inflammatorisk reaktion ved aktivering af IL-1b, IL-6 og IL-8 blev fundet. Herudover blev der set lokale forskelle i ekspressionsmønsteret afhængige af om stimuleringen var med DAMPS alene eller DAMPS / PAMPs kombination. Desuden understøtter fraværet af væsentlige forskelle i den respiratorisk burst aktivitet i hoved-nyreceller ideen om at det lokale respons på et vævsbeskadigelsen er det primære innate respons. Yderligere blev der fundet immunologiske effekter af intravenøs injektion af ß-glucan i huden. Således blev der observeret immunologiske forandringer i huden som tyder på en immunoglobulin switch-lignende proces med IgM / IgZ. Endvidere blev det fundet at iv. injektion med ß-glucan har en dosisafhængig effekt på stress niveauet i karper udtrykt som forøgelse af plasma cortisol niveau.Control of fish diseases is a great concern in aquaculture because of losses in the production. Drug choices for the treatment of common infectious diseases are becoming increasingly limited and expensive and, in some cases, unavailable due to the emergence of drug resistance in bacteria and fungi. This is why number of biological compounds, as an alternative to the drugs, has been used to reduce the risk of diseases and improve fish welfare by enhancement of non-specific defence system. Among them, ß-glucans, naturally occurring polysaccharides found in the cell wall of plants, bacteria and fungi, are some of the most powerful and extensively investigated immune modulants. ß-glucans have been proven to enhance the immune system and pharmacologically they are classified as biological response modifiers (BRM).The focus of the present thesis was on: 1. creation of a model for the examination of the biological impact of two commercially available ß-glucan enriched products on the wound healing process in common carp (Cyprinus carpio L.) in sterile, controlled conditions; 2. investigation of potential impact of intravenously injected ß-glucan on mucosal immune response and immunoglobulin switch-like process in common carp. In order to reach these objectives, different methods were used such as real-time quantitative PCR (RT-PCR) in order to measure the expression of immunerelated genes involved in wound healing process, ELISA for specific antibody detection, cortisol assay for measurement of stress level, respiratory burst assay for radical production measurement and image analysis.The results of this study showed that previous infections gave rise to changes regarding texture quality parameters in fresh fish meat, and were a starting point for use of immune modulators such as ß-glucans. Further work showed that bath in two commercially available ß-glucan enriched products, specifically MacroGard and 6.3 kDa oat fiber, had a direct positive effects on the wound closure in common carp and promoted faster wound healing compared to non-treated fish. We showed the immunological and regenerative response following stimulation with PAMPs and DAMPs in controlled conditions, without the exposure to pathogens, which resulted in an inflammatory response by activating IL-1b, IL-6 and IL-8. Local differences in expression pattern dependent on stimulation by DAMPs alone or DAMPs/PAMPs combination. In addition, the absence of marked differences on the respiratory burst activity in head-kidney cells supports the idea of a localized immune response to the site of injury. Due to direct and constant contact between skin and ß-glucan, bath treatment was an obvious choice to investigate. However, intravenous injection of ß-glucan showed it has a biological effect on skin as well. Once again we observed immunological changes taking place in skin of common carp, with IgM/IgZ immunoglobulin switch-like process. Finally, ß-glucan seems to work in dose-dependent manner, with elevating cortisol level when injected at high doses

Online Research Database In Technology

Mucosal immune response in common carp (Cyprinus carpio L.):Host pathogen interactions in relation to beta-glucan stimulation

http://orbit.dtu.dk/ws/files/51218909/Dominika_Przybylska_PhD_thesis..PDF

Mucosal immune response in common carp (Cyprinus carpio L.):Host pathogen interactions in relation to beta-glucan stimulation

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