Rainbow Trout (Oncorhynchus Mykiss) Intestinal Epithelial Cells as a Model for Studying Gut Immune Function and Effects of Functional Feed Ingredients

The objective of this study was to evaluate the suitability of the rainbow trout intestinal epithelial cell line (RTgutGC) as an in vitro model for studies of gut immune function and effects of functional feed ingredients. Effects of lipopolysaccharide (LPS) and three functional feed ingredients [nucleotides, mannanoligosaccharides (MOS), and beta-glucans] were evaluated in RTgutGC cells grown on conventional culture plates and transwell membranes. Permeation of fluorescently-labeled albumin, transepithelial electrical resistance (TEER), and tight junction protein expression confirmed the barrier function of the cells. Brush border membrane enzyme activities [leucine aminopeptidase (LAP) and maltase] were detected in the RTgutGC cells but activity levels were not modulated by any of the exposures. Immune related genes were expressed at comparable relative basal levels as these in rainbow trout distal intestine. LPS produced markedly elevated gene expression levels of the pro-inflammatory cytokines il1b, il6, il8, and tnfa but had no effect on ROS production. Immunostaining demonstrated increased F-actin contents after LPS exposure. Among the functional feed ingredients, MOS seemed to be the most potent modulator of RTgutGC immune and barrier function. MOS significantly increased albumin permeation and il1b, il6, il8, tnfa, and tgfb expression, but suppressed ROS production, cell proliferation and myd88 expression. Induced levels of il1b and il8 were also observed after treatment with nucleotides and beta-glucans. For barrier function related genes, all treatments up-regulated the expression of cldn3 and suppressed cdh1 levels. Beta-glucans increased TEER levels and F-actin content. Collectively, the present study has provided new information on how functional ingredients commonly applied in aquafeeds can affect intestinal epithelial function in fish. Our findings suggest that RTgutGC cells possess characteristic features of functional intestinal epithelial cells indicating a potential for use as an efficient in vitro model to evaluate effects of bioactive feed ingredients on gut immune and barrier functions and their underlying cellular mechanisms

The interplay between IPNV and host cell machinery : possible strategies for immune evasion

The aquatic birnaviruses cause diseases in different aquatic species worldwide. Infectious pancreatic necrosis (IPN), caused by IPN virus (IPNV) in salmonids, has arguably been the most economically important disease caused by these viruses. Although the number of cases and subsequently the impact of the disease has been reduced in recent years, concomitant with the use of genetically resistant fish, the ubiquitous nature of the virus and the lack of knowledge about reservoirs and interspecies transmission paves the way for disease reemergence in future. Understanding the virus interaction with host cell and the interplay with the immune system is crucial for development of viral therapies. Information about IPNV interaction with the host cells and immune system has been either lacking or inconsistent. In this thesis, we have attempted to understand some aspects of virus interaction with the host cell with the aim to unravel possible mechanisms used for immune evasion or subversion. Three studies that were conducted in vitro are reported. First, in paper I, we compared responses induced by different IPNV isolates. The study revealed that infection with different strains of IPNV results in different cytopathogenicity and cytokine profiles. Notably, The Sp serotype of IPNV had a higher capacity to suppress the expression of type I IFN and to inhibit protein synthesis. Consequently, in paper II and III, we focused our studies on a virulent Sp isolate. We selected common responses induced by all the isolates, namely inhibition of protein synthesis and apoptosis, and investigated the underlying mechanism and their impact on IPNV replication. The study reported in paper II was directed at understanding the roles these responses play during IPNV infection and their effect on virus replication. The findings indicate that inhibition of protein synthesis might be used to prevent the production of type I IFN and its antiviral effectors such as Mx. In addition, the mechanisms by which protein synthesis is inhibited was investigated partly paper II and III, and the involvement of eIF2α and PKR was demonstrated. However, paper III was mainly performed to address the interplay between IPNV and PKR using chemical inhibition. The data reported in this study shows that PKR activation is beneficial to the virus. Although, the detailed mechanisms involved could not be elucidated we speculate and provide suggestion about the possible underlying mechanisms. Collectively, the findings reported in this thesis provide new insights into IPNV interaction with host cells and suggest that IPNV is able to hijack some of the crucial antiviral responses and use it to evade type I IFN responses as well as to increase virus replication and facilitate its release from infected cells.Birnavirus er årsaken til sykdommer i forskjellige akvatiske arter over hele verden. Infeksiøs pankreasnekrose (IPN), forårsaket av IPN viruset (IPNV) hos laksefisk, har uten tvil vært den økonomisk mest viktige sykdommen relatert til disse virusene. Selv om antall sykdomstilfeller og betydningen av dem har blitt redusert i de siste årene, samtidig som genetisk motstandsdyktig fisk er tatt i bruk, så vil virusets utbredte natur kombinert med mangel på kunnskap om reservoarer og overføring mellom arter bane vei for reintroduksjon av sykdommen i fremtiden. Forståelse av virusets interaksjon med vertscellen og samspillet med immunsystemet er viktig for utvikling av virale behandlingsmetoder. Informasjon om IPNV sitt samspill med vertsceller og immunforsvaret har enten vært mangelfull eller inkonsistent. I denne oppgaven har vi forsøkt å forstå noen aspekter ved samspillet mellom virus og vertscelle med sikte på å avdekke mulige mekanismer viruset bruker for å unngå en immunreaksjon. Tre in vitro-baserte studier beskrives. I første artikkel (Paper I), sammenlignet vi immunresponser indusert av forskjellige akvatiske birnavirus-isolater. Studien viste at IPNV infeksjon resulterer i ulike cytopatogenitets- og cytokin-profiler. Særlig IPNV serotype Sp hadde en høyere kapasitet til å dempe ekspresjon av type I IFN og hemme proteinsyntese. Som en følge av dette, fokuserte vi våre studier på et virulent Sp isolat i de påfølgende artiklene (Paper II og III). Vi valgte vanlige responser indusert av alle isolater, dvs. hemming av proteinsyntesen og apoptose, og undersøkte underliggende mekanismer og deres innvirkning på IPNV replikering. Studien beskrevet i artikkel II var rettet mot å forstå responsenes roller i IPNV infeksjoner og deres effekt på virusreplikasjon. Funnene indikerer at inhibering av proteinsyntese kan bli brukt til å forhindre produksjon av type I IFN og dets antivirale effektorer som for eksempel Mx. I tillegg ble de mekanismene som fører til hemming av proteinsyntesen delvis undersøkt i artikkel II og III, hvor involvering av eIF2α og PKR ble demonstrert. Artikkel III var imidlertid hovedsakelig utført for å undersøke samspillet mellom IPNV og PKR ved bruk av kjemisk hemming. Dataene beskrevet i denne studien viser at PKR aktivering er gunstig for viruset. De detaljerte mekanismene som er involvert kunne ikke beskrives, men vi spekulerer og gi forslag om mulige underliggende mekanismer. Samlet gir resultatene som er beskrevet i denne oppgaven ny innsikt i IPNV sitt samspill med vertscellene og indikerer at IPNV er i stand til å kapre noen av de viktige antivirale responsene og bruke dette til å unngå type I IFN responser, samt til å øke virusreplikasjon og lette frigivelsen fra infiserte celler

Identification and characterization of two salmon louse heme peroxidases and their potential as vaccine antigens

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Apoptosis Induction by dsRNA-Dependent Protein Kinase R (PKR) in EPC Cells via Caspase 8 and 9 Pathways

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Efficacy and safety of boric acid as a preventive treatment against Saprolegnia infection in Nile tilapia (Oreochromis niloticus)

Saprolegniosis is a worldwide fungal-like infection affecting freshwater fishes and their eggs. Reports show high mortalities and subsequent economic losses annually from Saprolegnia infections. Most therapeutants against Saprolegnia spp. infections are inefficient and some have negative impact on the environment. In this study, we have investigated the ability of boric acid (BA) to prevent Saprolegnia infection in Nile tilapia (Oreochromis niloticus). BA inhibited radial growth of Saprolegnia hyphae in vitro. Complete in vitro growth inhibition was found at a concentration of ≥0.6 g/L. Inhibitory effects were also observed in vivo when Nile tilapia were experimentally challenged with Saprolegnia spores and followed over 10 days post challenge and under continuous exposure to different BA concentrations. No signs of saprolegniosis were observed in fish treated with BA at concentrations of 0.4 g/L and above. Comet assay revealed that BA has low toxicity in tilapia continuously exposed to concentrations of 0.2–0.6 g/L for 96 h. Additionally, no significant histomorphological changes were observed in BA-treated fish compared to non-treated controls. Alanine Aminotransferase (ALT) and Aspartate Aminotransferase (AST) enzyme levels indicated reduction in systemic tissue damage associated with Saprolegnia infection. This study demonstrates the potential of BA as a prophylactic measure against Saprolegnia infection in tilapia, and we recommend additional studies on environmental impact.publishedVersio

A whole-cell Lactococcus garvieae autovaccine protects Nile tilapia against infection

The autovaccine was produced in-house using a bacterial isolate from a diseased fish from the target farm. Three groups of 150 fish each were injected with either 1) an oil-adjuvanted, inactivated whole cell autovaccine, 2) adjuvant only or 3) PBS (negative control). Approximately 660 degree days post vaccination, the fish were challenged with 9x105 cfu bacteria/fish by intraperitoneal injection and monitored for a further 28 days. Protection against infections was measured by lack of/reduced bacterial loads both by bacterial re-isolation and immunohistochemistry as well as absence of clinical signs/pathology. Significantly less L. garvieae (p<0.03) was re-isolated from either the adjuvant only or control groups compared to the vaccinated group. Furthermore, a significantly high amount (p<0.001) of anti-L. garvieae specific antibodies were observed in the vaccinated group compared to the adjuvant only or control groups at time of challenge. This coincided with protection against infection measured by absence/reduced L. garvieae re-isolation from internal organs, reduced clinical signs and lack of pathology in this group. In the adjuvant only and control groups, bacteria were re-isolated from the kidney, liver, spleen, brain and eyes during the first 14 days. The findings suggest that oil-based vaccines can protect tilapia against L. garvieae infection through an antibody mediated response.publishedVersio

Efficacy and safety of boric acid as a preventive treatment against Saprolegnia infection in Nile tilapia (Oreochromis niloticus)

Saprolegniosis is a worldwide fungal-like infection affecting freshwater fishes and their eggs. Reports show high mortalities and subsequent economic losses annually from Saprolegnia infections. Most therapeutants against Saprolegnia spp. infections are inefficient and some have negative impact on the environment. In this study, we have investigated the ability of boric acid (BA) to prevent Saprolegnia infection in Nile tilapia (Oreochromis niloticus). BA inhibited radial growth of Saprolegnia hyphae in vitro. Complete in vitro growth inhibition was found at a concentration of ≥0.6 g/L. Inhibitory effects were also observed in vivo when Nile tilapia were experimentally challenged with Saprolegnia spores and followed over 10 days post challenge and under continuous exposure to different BA concentrations. No signs of saprolegniosis were observed in fish treated with BA at concentrations of 0.4 g/L and above. Comet assay revealed that BA has low toxicity in tilapia continuously exposed to concentrations of 0.2–0.6 g/L for 96 h. Additionally, no significant histomorphological changes were observed in BA-treated fish compared to non-treated controls. Alanine Aminotransferase (ALT) and Aspartate Aminotransferase (AST) enzyme levels indicated reduction in systemic tissue damage associated with Saprolegnia infection. This study demonstrates the potential of BA as a prophylactic measure against Saprolegnia infection in tilapia, and we recommend additional studies on environmental impact

cAMP induction post PGE2 treatment.

<p>Three cell lines, CHSE-214, RTG-2 and TO were used in this experiment. Cells were either treated with PGE2 (P) or left without treatment (C).</p

List of primers used for PCR and cloning.

<p>List of primers used for PCR and cloning.</p

Amino acid sequence of two EP4 variants in salmon.

<p>Comparison of amino acid sequence of two subtypes of EP4 gene identified in Atlantic salmon.</p