NUTRINFECT: New in vitro assays of fish pathogen infection across the intestinal epithelium and effects of functional feed ingredients

In aquaculture, the link between nutrition, immune responses and infection diseases is unclear. Knowledge about how feed components may affect mucosal infections is still unknown, not at least due to lack of targeted research tools. In vitro models may be used as targeted tools to study cell and tissue specific functions, and as screening tools to test new feed ingredients before feeding trials with live fish are run with the most promising candidates. While feeding trials demand the use of live fish and are expensive to run, in vitro models are cost effective and easy to run in the lab. The goal of the postdoc project NUTRINFECT was to establish new in vitro tools to evaluate effects of fish feed ingredients on infection diseases and gut health in salmonid fish. As pathogens including virus enter fish through mucosal surfaces found mainly in the gut, gills and skin, we wanted to establish infection models with important salmonid viruses by using an available gut cell line from rainbow trout (RTgutGC). By growing these gut cells on a porous membrane in a two-chamber well system, a coherent but permeable membrane is created simulating the intestinal epithelium. The first sub-goal of the project was to study infection in RTgutGC cells with infectious pancreas necrosis virus (IPNV), salmonid alpha virus (SAV) 3 and infectious salmon anemia virus (ISAV). The second sub-goal was to optimize the simulated gut environment by treating the cells with bile, either as total rainbow trout bile or as pure bile salts, since bile is known to have important functions in intestinal epithelium development and functionality. IPNV, SAV3 and ISAV infected and replicated in the RTgutGC cell line. The three viruses did however differ in infection courses and cellular responses. IPNV infection induced early cytopathogenic effect (CPE) (day1-2), meaning that the cells change structurally due to the virus, and the infection was swift reaching full CPE (cells totally destroyed) within one week. Increased infection dose with IPNV gave earlier CPE, increased virus replication in the cells and increased cellular anti-viral response. For SAV3, CPE was first visible somewhat later (day4) and the changes were less prominent, with a longer infection reaching full CPE after two weeks. For SAV3, increased infection dose gave earlier CPE and increased virus replication in the cells. However, the cellular anti-viral response against SAV3 was significantly reduced with increased dose, which may indicate that SAV3 are blocking cellular responses. For ISAV, CPE was visible at day4 and full CPE reached at day10 for low dose (moi=0.1), while high dose (moi=10) resulted in a delayed CPE. Low ISAV dose gave high virus replication in the cells, while viral replication after infection with high dose did not differ significantly from control cells. The anti-viral response against ISAV was equivalently high for low and high dose for several of the genes. This means that ISAV triggered responses in the RTgutGC cells both when virus replicated within the cells, but also when virus was present only in the surrounding medium without entering the cells. The gut barrier was significantly affected by all three viruses already 22 hours after infection, shown by increased barrier permeability. This in vitro model may thus be used further to study effects of feed components on infection with IPNV, SAV3 and ISAV in the salmonid gut. Bile salts are present in high amounts in the gut of live fish, but how bile salts may affect the functionality of gut cells except in lipid absorption has not been well studied. In salmonids, >90% of the bile salts are taurocholate. By treating the RTgutGC cells with bile, either as total bile taken from the rainbow trout gall bladder or as pure taurocholate, gene expression analyses revealed significant changes in essential gut metabolism and function genes without compromising the cellular viability. High levels of bile salts (>0.5 mg/mL) were however toxic for the cells. RNA sequencing analyses showed that four days treatment with bile salt significantly changed the transcriptome of the RTgutGC cells. Especially genes related to lipid metabolism, retinoic acid metabolism (linked to vitamin A function), immune functions and transport were affected. More targeted analyses with qPCR showed that cell barrier functions and brush border membrane enzymes were affected, which indicate a function for bile salts in maturation of the gut cells. Bile salt should thus be included in the cultivating medium for gut cells in in vitro studies of gut cell functions, to better mimic the gut environment in live fish. Treatment with rainbow trout bile and pure taurocholate resulted however in somewhat differing effects, and the pure taurocholate gave more responses in the RTgutGC cells. This indicates that other bile components modulated the response in addition to the taurocholate. Effects from these components should thus be further investigated.

Prosjektet har resultert i at vi har etablert in vitro smittemodeller med viktige laksevirus i en tarmcellelinje fra regnbueørret. Disse smittemodellene vil brukes videre av vår forskningsgruppe for å undersøke effekter av nye fôringredienser for infeksjonssykdommer og tarmhelse hos laksefisk. Vi har allerede pågående prosjekter hvor vi drar nytte av denne kunnskapen (blant annet EU Horizon 2020-prosjekt) og vi vil søke nye prosjekter for å bygge videre på det som ble etablert i postdok-prosjektet. Videre har prosjektet ført til at vi har endret vekstvilkårene for tarmceller in vitro ved å tilsette gallesalter for å bedre mimikere det naturlige miljøet i laksetarm, ettersom gallen hadde signifikant betydning for tarmcellenes utvikling og modning. Denne kunnskapen er allerede direkte tatt i bruk i andre pågående prosjekter. Prosjektet har videre ført til at postdok-stipendiaten har tilegnet seg betydelig økt kompetanse blant annet innen områdene cellekultur, infeksjonsbiologi og gallemetabolisme, samt erfaring i prosjektledelse. Postdok-stipendiaten vil derfor, inn i sin neste stilling, være en økt ressurs for forskningsgruppen. Resultatene fra dette postdok-arbeidet vil kunne forenkle fremtidig evaluering av stadig mer komplekse fôr innen akvakultur, for å predikere effekter på tarmhelse og sykdomsresistens hos fisk, og for å identifisere bioaktive ingredienser som kan ha negative eller positive effekter for dyret. Ved bruk av disse in vitro smittemodellene som kartleggingsverktøy og for å studere isolerte celle- og vevs-funksjoner, kan dessuten antall levende fisk som må ofres reduseres. Arbeidet vil dermed bidra til å sikre optimal tarmhelse hos laksefisk og optimal bruk av bærekraftige fôringredienser, samt bidra til reduksjon av levende fisk som benyttes i forskning.

Present knowledge basis for understanding relationships between nutrition, immune response and production diseases in aquaculture is very limited, not at least due to lack of targeted research tools. The goal of this post-doctoral research fellowship is to employ two established in vitro salmonid intestinal barrier models (the RTgutGC cell line and Ussing chamber technology) as screening systems for predicting effects of novel fish feed ingredients on fish pathogen infection, antigen translocation across the intestine and gut health. Impact on the epithelial barrier function and cell responses will particulary be studied. The proposed work will be closely linked to several currently running research projects (Foods of Norway (RCN), PROMAC (RCN), ArticFunc (RCN/RFFNORD) and GutMatters (FHF)), all with a focus on novel feed ingredients to salmon, functional feed additives and gut health. Scientific achievements from the work will simplify the future screening of increasingly more complex aquafeeds, in order to predict effects on fish gut health and disease resistance, and to identify bioactive compounds having negative or positive effects on the animal. The work will aid in securing optimal fish gut function and healthy fish tolerant to changes in production conditions, and optimal use of feed ingredients from sustainable resources. Thus, the present work will generate knowledge vital for achieving economical and sustainable growth in Norwegian aquaculture. The applicate project meets the call for tools that will contribute to replacement and reduction of live animals in fish research. The project will provide recruitment of a young academic working in an internationally well recognized research group.