Dafinet poster november 2010

NOVEMBER 9 & 10, 2010 IMMUNE RESPONSES IN FISH: FROM GENE TO FUNCTION
Invited speakers:
Dr. Barbara Nowak, Australia
University of Copenhagen
Dr. Bertrand Collet, Scotland
Faculty of Life Sciences
Dr. Simon Jones, Canada

Book of abstracts
DAFINET November 9 and 10, 2010
University of Copenhagen, Denmark
DAFINET is supported by the Danish Council for Strategic Research
The book of abstracts is edited by
Kurt Buchmann, Per W. Kania and Lars Holten-Andersen
Printed by
Frederiksberg Bogtrykkeri 2010

Danish Fish Immunology Research Centre
and Network
Two day workshop on:
Immune Responses in Fish: From Gene to Function
Date: November 9 and 10, 2010
November 9 from 13.00-17.00 (1 to 5 pm)
November 10 from 10.00-16.00 (10 am to 4 pm)
Lecture Theatre 1-01
University of Copenhagen
Faculty of Life Sciences
Bülowsvej 17
DK-1870 Frederiksberg C

Danish Fish Immunology Research Centre
and Network
Two day workshop on:
Immune Responses in Fish: From Gene to Function
PROGRAM

November 9, 2010
Pre-workshop activities 10.00-13.00
DAFINET board meeting for board members and associates (lecture theatre) Lunch for all participants (Stigbøjlen 7) Workshop start in lecture theatre
13.00 Kurt Buchmann, University of Copenhagen
Opening and welcome address by the DAFINET leader 13.15 Barbara Nowak, University of Tasmania, Tasmania, Australia
Fish immunology research Down Under: Vaccination against Yersiniosis 14.00 Coffee break
14.30 Simon Jones, Pacific Biological Station, Nanaimo, British Columbia, Canada
Innate immunity to the salmon louse Lepeophtheirus salmonis among salmonids 15.15 Discussion break
15.30 Bertrand Collet, Marine Scotland, Marine Laboratory, Aberdeen, Scotland
Interactions between virus and hosts: In vitro model studies 16.15 Discussion break
16.30 Jiwan Kumar Chettri, University of Copenhagen, Faculty of Life Sciences,
Frederiksberg, Denmark
PAMP induced expression of immune relevant genes in head kidney leukocytes of rainbow trout (Oncorhynchus mykiss) 17.00 Closing of the session
November 10, 2010
10.00 Jakob Skov, University of Copenhagen, Faculty of Life Sciences,
Frederiksberg, Denmark
Effects of β-glucan in feed on lysozyme activity and antibody response in vaccinated and unvaccinated rainbow trout (Oncorhynchus mykiss) 10.15 Alf Skovgaard, University of Copenhagen, Faculty of Life Sciences,
Frederiksberg, Denmark
PKD-infections in Wild and Farmed Trout – Occurrence of Tetracapsuloides in Danish 10.30 Mikkel-Ole Skjødt, University of Southern Denmark
Identification of a novel plasma protein that regulates innate immune functions 11.00 Coffee break
11.30 Moonika M. Olsen, University of Copenhagen, Faculty of Life Sciences,
Frederiksberg, Denmark
Putative T-cells and IgT involved in the response of rainbow trout gills to Ichthyophthirius multifiliis infections: molecular and immunohistochemical studies 12.00 Lunch (Stigbøjlen 7)
13.00 Barbara Nowak, University of Tasmania, Tasmania, Australia
Striped trumpeter health in hatchery 13.30 Kasper R. Villumsen, University of Copenhagen, Frederiksberg, Denmark
Comparative resistance towards infection with Y. ruckeri in vaccinated and non- vaccinated rainbow trout 14.00 Coffee break
14.30 Ellen Lorenzen, Technical University of Denmark, National Veterinary
Laboratory, Århus, Denmark
Experimental vaccination of small turbot against bacterial and viral pathogens. 15.00 Børge Nilsen Fredriksen, University of Tromsø, Norway
Correlates of protection for infectious pancreatic necrosis virus in Atlantic salmon (Salmo salar) vaccinated with oil-based inactivated vaccines. 15.30 Hans-Christian Ingerslev, Technical University of Denmark, National Food
Institute, Lyngby, Denmark
Fish quality – linking previous infections to the quality of consumers' fillet 16.00 Closing of the workshop

ABSTRACTS

Welcome address by the DAFINET leader
Kurt Buchmann
University of Copenhagen, Faculty of Life Sciences
Department of Veterinary Disease Biology, Frederiksberg C, Denmark

The Danish Fish Immunology Research Centre and Network DAFINET, to be visited at
, established in January 2009, is soon ready to celebrate the finalization of the first two years of work. The main research topic within the consortium is to explore the ontogenetic development of the fish immune system with special emphasis on rainbow trout. This salmonid fish is currently being produced around the world ranging from China in the Far East, to North America in the west, Australia and New Zealand in the South and Norway in the North. The total annual production amounts to more than 600,000 metric tonnes and is thereby one of the main actors in the still rapidly increasing aquaculture production. The species represents not only an important food product but is also a valuable model within fish immunology. However, researchers at the centre will perform comparison with at least a number of the already described 28,000 teleosts species. One of the expected results from the research effort is a basic knowledge of how early fish can be afforded protection against various diseases. This will facilitate targeted vaccination and immunostimulation of even very young fish stages and thereby reduction of drug application. A second outcome is the production of a range of molecular tools to be used in basic and applied research within fish immunology. The consortium behind the centre comprises 15 partners from a series of research institutions and private companies located not only in Denmark but also in Scotland, Norway and Germany. The centre has an external advisory board headed by research director Scott LaPatra from Idaho, USA. During this two-day workshop some of the latest results form the centre-activities will be presented. In addition, the centre has called in some outstanding experts from Canada, Australia and Scotland in order to place the local research in a broader framework and stimulate international collaboration. Innate immunity to the salmon louse Lepeophtheirus salmonis among
salmonids
Simon R.M. Jones
Pacific Biological Station, Fisheries and Oceans Canada, 3190 Hammond Bay Road,
Nanaimo, British Columbia, V9T 6N7, Canada
The salmon louse Lepeophtheirus salmonis incurs significant costs to salmon mariculture
in the north Atlantic and Pacific Oceans. In addition, there is the potential for infections to spill-back and cause harm to wild salmon. We utilized a controlled laboratory exposure model to explore host – parasite interactions and specifically to characterize resistance to the parasite among Pacific and Atlantic salmon. Significantly lower levels of infection were observed on juvenile pink salmon (Oncorhynchus gorbuscha) compared with chum (O. keta) or Atlantic salmon (Salmo salar) following exposure to infective copepodids. Parasites were quickly rejected from pink salmon whereas they persisted on chum and Atlantic salmon until they had molted to larger more aggressive stages. Anaemia and elevated serum cortisol and serum prostaglandin E2 were associated with infections on chum and Atlantic salmon but not on pink salmon. Pink salmon possess an innate immunity to the parasite that first develops in salmon weighing less than 1 g and functions despite feed deprivation. Gene expression, measured by using quantitative RT- PCR, showed that between 7 and 21 days after exposure IL-1β and IL-8 were upregulated in the skin and TNFα was upregulated in the head kidney of pink, but not chum salmon, suggesting localized inflammation is a mechanism for early parasite rejection in pink salmon and that a systemic response is also involved. Very little of the cutaneous transcriptomic response was shown to be caused by mechanical abrasion, suggesting that other attributes of the parasite were responsible for triggering the reaction. The onset of innate immunity in pink salmon was explored by using microarray and qRT-PCR to profile the transcriptome of pink salmon belonging to susceptible (0.3g), intermediate (0.7g) and resistant (2.4g) size classes. Transcripts in susceptible and intermediate size classes favored cell mobility, wound healing and immunity. In contrast, transcripts in susceptible 0.3g fish favored systemic stressors, inhibition of cell proliferation and inflammation. The curious divergence in natural resistance to L. salmonis observed between two morphologically similar and sympatric species of Pacific salmon is discussed in the context of life history strategies. Fish immunology research Down Under: Vaccination against
Yersiniosis
Barbara F. Nowak
National Centre for Marine Conservation and Resource Sustainability, AMC, University
of Tasmania, Launceston, Tasmania, Australia
Our fish immunology research focuses on commercial fish species farmed and ranched in
Australia. Atlantic salmon, southern bluefin tuna and striped trumpeter are the main species investigated by our group. With regard to Atlantic salmon main areas of interest are immunomodulation, including improvement of existing vaccines such as Yersinivac B, which is used against yersiniosis. Yersiniosis killed half a million fish in just one hatchery during a 6 month period during 2007, resulting in a smolt shortfall for the entire Tasmanian salmon industry. Australian isolate serovar O1b is pathogenic to Atlantic salmon but not rainbow trout. While most Tasmanian Atlantic salmon are now (since 2006) vaccinated against yersiniosis (Yersinivac-B, developed by DPIPWE Tasmania, manufactured by Intervet SP), significant disease outbreaks still occur. Our research focused on effects of different application strategies. We showed that the application used by the industry was ineffective and that double dip was better than single dip or bath vaccination. The industry has not adopted double dip application as their strategy for Yersinivac-B. Oral vaccination showed promising results in a series of challenges. Even 38 weeks post vaccination (23 weeks since last oral boost) the fish which were double dip vaccinated and received oral boost showed no mortalities in a challenge, low mortalities were present in the double dip vaccinated and oral vaccinated groups, whereas the greatest mortalities were present in the unvaccinated fish. There were significant differences in gene expression between vaccinated and unvaccinated fish, particularly during challenge. Antimicrobial peptides which were differentially expressed were investigated further to elucidate their role in protection against infection with Yersinia ruckeri. Striped trumpeter health in hatchery
Barbara F. Nowak
National Centre for Marine Conservation and Resource Sustainability, AMC, University
of Tasmania, Launceston, Tasmania, Australia
Striped trumpeter Latris lineata is a temperate marine fish species which has been
investigated for aquaculture in Tasmania. In the last few years we investigated health of striped trumpeter, in particular its immune response, parasitic infections, epitheliocystis and development of urinary calculi in the larvae. A partial sequence of the recombination activating gene-1 (RAG-1) and the full sequence of the immunoglubulin M (IgM) heavy chain were discovered in the striped trumpeter (Latris lineata). Both genes showed good homology to other vertebrate sequences. The expression of the two genes was followed throughout the early developmental stages of the larvae (5-100 dph) and used as a marker for ontogeny of the adaptive immune response. Using RT-PCR, RAG-1 expression was detectable at 5 dph and increased steadily until 80 dph before becoming undetectable at 100 dph. IgM expression was also detectable at 5 dph, but decreased at 50 dph before increasing again at 80 and 100 dph. These patterns of expression suggest that the striped trumpeter possess mature B cells with surface IgM at 100 dph. However, immunological competence is likely not reached until a later date. Urinary calculi are mineral deposits present in most marine fish larvae reared in hatcheries. The calculi were detected inside the urinary bladder of the striped trumpeter larvae on 7 days post hatch, which was the day after the larvae had started to feed on enriched rotifers. While there was an obvious pathology caused by the calculi, there was no relationship between the presence of calculi and survival or condition of the larvae. It appeared that the availability of food could influence prevalence of calculi, with larvae on restricted diet having higher prevalence. Most infections occur after the fish are moved to grow out tanks where water is less treated than in the hatchery. Epitheliocystis was present in many juvenile cohorts, sometimes it was severe. The causative agent was partially identified using PCR and its sequences was novel, sharing only a distant 83% sequence similarity to the next closest 16S rDNA sequences from Candidatus Piscichlamydia salmonis isolates. Average prevalence of epitheliocystis was 73% and it was affected by cohort and season. There was a significant relationship between severity of epitheliocystis and lysozyme activity. The prevalence and intensity of parasitic infections was highly variable between cohorts Interactions between virus and hosts: In vitro model studies.
1Katherine Lester, 1Katy Urquhart, 1Malcolm Hall, 2Suresh Gahlawat,
1David Smail, 1Bertrand Collet.
1 Marine Scotland, Marine Laboratory, Aberdeen, UK 2 CCS Haryana Agricultural University, Hisar, India Interferons (IFN) are cytokines that are involved in the defence against viral infections. Type I IFN includes many different subtypes and is responsible for the innate early antiviral mechanisms. Cells respond to IFN by producing a large number of molecules with direct or indirect antiviral properties. Among those, the Mx protein has been initially described as conferring resistance to viruses of the Orthomyxoviridae influenza virus. Although the basis of its antiviral property is not fully understood, it is believed that its accumulation in the cells interferes with the viral protein trafficking and particle packaging resulting in a general inhibition in the viral propagation. In fish, the Mx protein and its coding gene have been used as a marker of type I IFN activity for many years. However, the inherent contribution of the Mx protein to viral resistance and its spectrum is unknown. We describe here the isolation of two double-recombinant chinook salmon embryo clones CHSE-TOF5-MX8 and CHSE- TOF5-MX10 that express the rainbow trout Mx1 under the control of doxycycline (DOX) as part of the inducible expression Tet-Off system. Representatives of 4 categories of viruses, based on their genome, causing serious damage to the global salmonid farming industry were used in this study: positive-sense single-stranded RNA (salmonid alphavirus - SAV), negative-sense single-stranded RNA (infectious haematopoeitic necrosis virus - IHNV), double-stranded RNA (infectious pancreatic necrosis virus – IPNV) and double-stranded DNA (epizootic haematopoietic necrosis virus - EHNV). They were tested and compared for i) their ability to induce the Mx gene in the RTG-P1 reporter cell line and ii) their sensitivity to accumulation of Mx protein in the cytoplasm in the newly engineered CHSE-TOF5-MX clones. This result gives clear evidence that distinct groups of viruses and distinct isolates of the same virus have evolved different strategies to circumvent the antiviral effect of IFN. PAMP induced expression of immune relevant genes in head kidney
leukocytes of rainbow trout (Oncorhynchus mykiss)
Jiwan K. Chettri, Martin K. Raida, Lars Holten-Andersen, Per W. Kania
and Kurt Buchmann
University of Copenhagen, Faculty of Life Sciences, Department of Veterinary Disease Biology, Frederiksberg C, Denmark Host immune responses elicited by invading pathogens depend on recognition of the pathogen by specific receptors present on phagocytic cells. However, the reactions to viral, bacterial, parasitic and fungal pathogens vary according to the pathogen-associated molecular patterns (PAMPs) on the surface of the invader. Phagocytic cells are known to initiate a respiratory burst following an exposure to the pathogen, but the underlying and associated specific elements are poorly elucidated in fish. The present study describes the differential response of head kidney leukocytes from rainbow trout (Oncorhynchus mykiss) to different pathogen associated molecular patterns mimicking viral (poly I:C), bacterial (flagellin and LPS) and fungal infections (zymosan and β-glucan). Transcript of cytokines related to inflammation (IL-1β, IL-6, IL-10 and TNF-α) were highly up- regulated following LPS exposure whereas flagellin or poly I:C induced merely moderate reactions. In contrast, IFN-γ expression was significantly higher in the poly I:C stimulated group compared to LPS group. When head kidney cells were exposed to zymosan or β-glucan, genes encoding IL-1β, TNF-α, IL-6 and IL-10 became up- regulated. Their level of up-regulation was comparable to LPS but the kinetics differed. In particular, TNF-α induction was considerably slower when stimulated with zymosan or β-glucan. The gene encoding COX-2 enzyme, which is a central element in initiation of inflammatory reactions, was significantly higher in stimulated cells but a depressing effect of high concentrations of LPS and zymosan became evident after 4 h exposure. This study suggests that rainbow trout leukocytes respond differently to viral, bacterial and fungal PAMPs, which may reflect activation of specific signaling cascades eventually leading to activation of different immune effector molecules.
Effects of β-glucan in feed on lysozyme activity and antibody response
in vaccinated and unvaccinated rainbow trout (Oncorhynchus mykiss)
Jakob Skov
University of Copenhagen, Faculty of Life Sciences, Department of Veterinary Disease
Biology, Laboratory of Aquatic Pathobiology, Stigbøjlen 7, 1870 Frederiksberg C.

The potential immunostimulatory effect of dietary β-glucan in rainbow trout was
investigated by including 1% β-1,3-glucan (paramylon from Euglena gracilis) in the fish feed for 12 weeks of continuous feeding. Four different experimental groups (no β- glucan/no vaccination; β-glucan/no vaccination; no β-glucan/vaccination and β- glucan/vaccination) were examined in duplicate. Fish were bath-vaccinated using a commercial Y. ruckeri bacterin after two weeks of feeding and bath-challenged with a predetermined LD50 of live Y. ruckeri serotype 01 six weeks post vaccination. Mortality was assessed during 4 weeks post-challenge. Survival following challenge showed no effect of the β-glucan among unvaccinated (Logrank Test; p = 0.97) or vaccinated fish (p = 0.99). A cumulative percent mortality (CPM) of 16.7% was induced in the unvaccinated groups following challenge opposed to 2.5% CPM in the vaccinated groups. However, a significant difference in survival was found between unvaccinated and vaccinated fish (p = 0.0002) irrespective of the β-glucan feeding indicating a positive effect of the vaccine. Blood was sampled at different time points before and after the vaccination and challenge events to assess lysozyme activity (turbidimetric assay) and presence of Y. ruckeri-specific antibodies (ELISA). Data from lysozyme and antibody analyses will be presented. PKD-infections in Wild and Farmed Trout – Occurrence of
Tetracapsuloides in Danish Streams
Alf Skovgaard and Kurt Buchmann
University of Copenhagen, Faculty of Life Sciences, Laboratory of Aquatic
Pathobiology, Department of Veterinary Disease Biology, Frederiksberg, Denmark

Proliferative kidney disease (PKD) is caused by infection with the parasitic myxozoan
Tetracapsuloides bryosalmonae. The disease is common in salmonid fishes in Europe and North America and is responsible for considerable economic losses to freshwater aquaculture of salmonids in these regions. Infection with T. bryosalmonae is not transmitted horizontally from fish to fish, but has a life cycle that includes different species of freshwater bryozoans. PKD is also believed to have severe effects on populations of wild salmonids, exemplified by reports of high mortality in juvenile Salmo salar in Norway and S. trutta in Swiss rivers. However, the occurrence and effect of T. bryosalmonae in nature has not been explored in much detail despite the fact that infection in fish farms has its origin from naturally occurring hosts. We here report on the occurrence of T. bryosalmonae in young-of–the-year salmonids from Danish streams and river tributaries in Zealand, Funen and Jutland.
Putative T-cells and IgT involved in the response of rainbow trout gills
to Ichthyophthirius multifiliis infections: molecular and
immunohistochemical studies
Moonika M. Olsen, Per W. Kania, Rasmus D. Heinecke, Karsten Skjoedt,
Kamilla Rasmussen, Kurt Buchmann
University of Copenhagen, Faculty of Life Sciences, Department of Veterinary Disease
Biology, Laboratory of Aquatic Pathobiology, Stigbøjlen 7, DK-1870 Frederiksberg C,
Denmark
Department of Cancer and Inflammation, Institute of Molecular Medicine, University of
Southern Denmark, Winsløwparken 21.1, DK-5000 Odense, Denmark

The parasitic ciliate Ichthyophthirius multifiliis infecting skin, fins and gills of fish
induces a protective immune response in rainbow trout (Oncorhynchus mykiss) surviving the infection and a similar protection can be conferred by i.p. injection of live theronts. A combined molecular and immunohistochemical approach has been used in this work for pinpointing cellular and humoral immune factors in gill tissue involved in the response. Fish were immunized by intra-peritoneal injection of live I. multifiliis theronts, control fish were injected with PBS and subgroups were treated with the immuno-suppressant hydrocortisone before fish were challenged with live theronts. Significant up-regulations of genes encoding IgM, IgT, C3, SAA, IFN-γ, IL-8 and IL-22 were induced by immunization and challenge. Hydrocortisone treatment had a significant down-regulating effect on genes incoding IgT, CD4 and CD8, IFN-γ, IL-8 and IL-22 in all groups. Immunohistochemistry, using monoclonal antibodies to detect cellular markers, demonstrated active involvement of CD8, MHC II, IgT and IgM positive cells in gill tissue. Putative T-cells (CD8 positive cells) were detected in the intraepithelial lymphoid tissue located at the base of primary gill filaments and in hyperplastic gill tissue but following challenge a clear efflux of these cells was detected. MHC II positive cells were distributed across the primary and secondary gill lamellae and accumulated in hyperplastic tissue but hydrocortisone treatment affected their density negatively in both immunized and non-immunized fish. IgT positive cells were present in the epithelial lining of the gill filaments and lamellae (suggesting a primary role of this protein in the mucosal defence against the ciliate) whereas IgM positive cells were found only in arteries and the lamellar arterioles. The present work indicates an intensive activity and specialized function of immune cells (B-cells, T-cells and macrophages) interacting with immunoglobulins IgT and IgM and orchestrated by cytokines in gill tissue reacting against I. multifiliis. Identification of a novel plasma protein that regulates innate immune
functions
Mikkel-Ole Skjødt, Tina Hummelshøj, Yaseelan Palarasah, Christian
Honore, Claus Koch, Karsten Skjødt and Peter Garred
Department of Cancer and Inflammation, Institute of Molecular Medicine, University of
Southern Denmark, Odense, Denmark
Laboratory of Molecular Medicine, Department of Clinical Immunology, Rigshospitalet,
Copenhagen, Denmark
The complement system is a vital part of the innate immune system and plays an essential
role of in the protection against invading pathogens. On the other hand uncontrolled activation of complement is fatal and leads to tissue damage and systemic inflammatory responses in a range of different diseases. The lectin complement pathway involves circulating complexes consisting of mannose-binding lectin (MBL) or Ficolins in association with the serine proteases MASP-1, -2 and -3. We have identified a novel plasma protein of 45kDa associated with MBL and Ficolin. We named this protein MBL/Ficolin associated protein-1 or MAP-1. MAP-1 is generated by differential splicing of the MASP1 gene and contains exon 1-8 and a novel exon encoding an in-frame stop codon. The corresponding protein lacks the serine protease domain but includes most of the heavy chain of MASP-1/-3. Additionally MAP-1 contains 17 unique C-terminal amino acids. By use of real-time RQ-PCR and immunohistochemistry we found that MAP-1 is highly expressed in myocardial and skeletal muscle tissues and to some degree in liver and neural tissues with a different expression profile from that observed for MASP-1 and MASP-3. MAP-1 co-precipitated from human serum with MBL, Ficolin-2 and Ficolin-3 and was able to inhibit complement activation via both the Ficolin and MBL pathway. In conclusion we have identified a novel 45kDa plasma protein derived from the MASP1 gene, which is highly expressed in striated muscle tissues. It is found in complex with MBL and Ficolin and may function as a potent local or systemic inhibitor of complement mediated inflammation in vivo. Comparative resistance towards infection with Y. ruckeri in vaccinated
and non-vaccinated rainbow trout
Kasper Rømer Villumsen & Martin Kristian Raida
Laboratory of Aquatic Pathobiology, Department of Veterinary Disease Biology, Section
of Biomedicine, Faculty of Life Sciences, University of Copenhagen, Denmark

The causative bacterial agent for enteric red mouth disease, Yersinia ruckeri, presents a
valuable target for development of efficient vaccines. Since valuable lessons can be learned from the investigation of host-pathogen interactions during infection, the focus of the present study is to investigate differences in the immune responses as well as infection levels between non-vaccinated control fish and fish immersion-vaccinated with formalin-killed, GFP-tagged Y. ruckeri (1x109 CFU/ml, 10 minutes, serotype O1, biotype 2). Seven months post-vaccination, both groups were bath-challenged with LD50-doses of Y. ruckeri (serotype O1, biotype 1 and 2), after which fish mortality was recorded and individual fish were sampled for both Real-Time Quantitative PCR (RT-qPCR) as well as immunohistochemical analysis. Challenge with biotype 2 resulted in very low mortalities with no significant difference in mortality between vaccinated and non-vaccinated fish. Challenge with biotype 1 resulted in a 35% mortality rate in the non-vaccinated fish. The mortality in the vaccinated group was significantly decreased (P=0.0001). At 3 days post challenge with biotype 1, 80% of non-vaccinated and 60% of vaccinated fish were shown to be infected with Y. ruckeri by RT-qPCR analysis. However, at 7 days post-challenge, while RT-qPCR results showed that 40% of non-vaccinated fish were still infected, no infection was found in vaccinated fish. This corresponds well with the difference in mortality rates between the two groups. Furthermore, initial mortalities in the non- vaccinated group are seen 7 days post challenge, increasing within 5 days from that point. This also corresponds well with the level of infection in each group at day 7 post- challenge. Immunohistochemical analysis of non-vaccinated and vaccinated fish sampled 3 and 7 days after challenge with biotype 1, shows results similar to the RT-qPCR results. Using polyclonal rabbit antibodies against Y. ruckeri, minor points of infection are seen in tissues of non-vaccinated fish after 3 days, and after 7 days massive infections are present in several tissues. Minor infections are found in the vaccinated fish after both 3 and 7 days, but to a smaller extend than the ones found in the non-vaccinated group. The results so far, thus indicate that the survival of the vaccinated fish after bacterial challenge seems to be correlated with an ability to clear bacterial infection over time. Additionally, the results indicate that immersion vaccines based on Y. ruckeri serotype O1, biotype 2 confers significant cross-protection against biotype 1, indicating potential importance of antibodies in resistance towards infection with Y. ruckeri. We are currently running an experiment involving passive transfer of immunity, involving transfer of serum from immunized to naïve rainbow trout, in order to further understand the role of antibodies.

Experimental vaccination of small turbot against bacterial and viral
pathogens.
Ellen Lorenzen1, Jesper S. Rasmussen1, Torben E. Kjær1, Katja Einer-
Jensen1, Kirsten Engell-Sørensen2, Inger Dalsgaard1, Jørgen Nylén3, Kurt
Buchmann4 and Niels Lorenzen1.
1National Veterinary Institute, Technical University of Denmark 2 Fishlab, 3 Intervet Schering Plough, Animal Health, 4 University of Copenhagen, Faculty of Life Sciences Turbot (Scophthalmus maximus) is a highly estimated fish species within aquaculture due to its delicious and tasty meet. As with other fish species, turbot occasionally suffers from infectious diseases when reared under intensive farming conditions. Two pathogens, naturally occurring in seawater, have caused major problems in cultured turbot, namely the Gram negative bacterium Vibrio anguillarum and the RNA-virus viral haemorrhagic septicaemia virus (VHSV). V. anguillarum is primarily a problem at the early fry/fingerling size stages, while VHSV can cause problems at all life stages. The present studies were undertaken as a part of the DAFINET collaboration to analyse the protective effect of immersion vaccination against vibriosis and injection vaccination against VHS. Turbot fry were vaccinated by immersion in commercial vaccines against A. salmonicida and V. anguillarum one, two or five times prior to challenge. Challenge was performed by immersion in a suspension of V.anguillarum bacteria. The results showed that on average, fish vaccinated more than once and last time only 5 weeks prior to challenge performed better compared to fish vaccinated only once and 12-13 weeks prior to challenge. For VHSV, the fish (approx. 5 g each) were vaccinated by intramuscular injection with a DNA-vaccine against VHSV. Challenge was performed by immersion in - or by ip-injection of VHSV 8 weeks post vaccination. The vhs-DNA vaccine protected the fish against VHSV independently of the challenge method and the results suggested that protection was mediated by specific immune mechanisms such as virus-neutralizing
Correlates of protection for infectious pancreatic necrosis virus in
Atlantic salmon (Salmo salar) vaccinated with oil-based inactivated
vaccines
BN. Fredriksen2, Hetron M. Munang'andu1, S. Mutoloki1, R. Dalmo2 and
Ø. Evensen1.
1Norwegian School of Veterinary Science, PO Box 8146, N-0033 Oslo, Norway. 2University of Tromsø, Faculty of Biosciences, Fisheries & Economics, 9037 Tromsø, Norway. Vaccination has been an important disease control strategy in the aquaculture industry for more than two decades. Despite the success of some of the commercial vaccines currently used in aquaculture, there is little information on correlates of immune protection in fish vaccinology. In the present study we investigated the correlates of protection in Atlantic salmon immunized against infectious pancreatic necrosis virus (IPNV). Two oil-based inactivated vaccines were prepared and administered intraperitoneally as a high-antigen- dose (HiAg) and low-antigen-dose (LoAg) vaccine using the highly virulent Norwegian Sp strain rNVI015-TA made by reverse genetics. Eight weeks post-vaccination, fish were challenged by cohabitation with virus shedders. Sampling was carried out at days 28 & 56 post-vaccination, and at 7, 10, 14, 21, 56 and 117 days post challenge. Samples collected included blood, head kidney, spleen, liver and pancreas. Antibody responses were evaluated using ELISA while qPCR was used to measure the quantity of virus in the infected organs after challenge. The variables used to measure the correlates of protection included (i) comparison of antibody responses of HiAg and LoAg dose groups, (ii) ability of the generated antibody responses in reducing post challenge mortality, (iii) ability of the generated antibodies to limit virus distribution and, (iv) ability to reduce viral loads in the infected organs during the pre-clinical and clinical stages of infection. The highest protection (90% RPS) was in the group vaccinated with the HiAg vaccine group which also had the highest antibody levels at challenge. The LoAg dose group (43% RPS) had low antibody levels at challenge. Post challenge virus distribution correlated with pre- challenge antibody levels with most fish (>70%) in the unvaccinated control group having virus in the head kidney, pancreas and spleen during the pre-clinical period while only a few fish (<10%) in the vaccinated groups had virus. Increase in viral loads was linked to the reduction in antibody levels at the peak of the infection (day 21 post challenge) with fish in the HiAg dose group having low viral loads with relatively high antibody levels compared to the LoAg group which had high viral loads and no antibodies detected during the acute stage of infection. Overall, our findings demonstrate that virus neutralization by antibodies is an important mechanism by which protection against IPNV is achieved as it reduces post challenge mortality, limits virus distribution and reduces viral load in the infected tissues.
Fish quality – linking previous infections to the quality of consumers'
fillet
Hans-Christian Ingerslev and Michael Engelbrecht Nielsen
National Food Institute, Division of Industrial Food Research, Section for Biological
Quality. Lyngby, Denmark

The quality of the fish meat is dependent upon a wide range of biological and non-
biological factors. In the present study it has been established that previous infections by Vibrio anguillarum in rainbow trout (Oncorhynchus mykiss) influence the quality of the fish meat (fillet) at slaughter more than after the fish have recovered from the infection. The texture of the fillet analysed by sensory analysis showed changes, which could be explained by previous tissue damage caused by the infection. These changes indicated formation of scars in affected tissue during the processes of tissue repair, which gave rise to a more fibrous, tougher and flaky texture of the fillets. List of participants
First
Surname E-mail
Affilation
Department of Veterinary Disease Biology, University of Copenhagen, Denmark Department of Veterinary Disease Biology, University of Copenhagen, Denmark Læskovvej 235, 4632 Bjæverskov, Denmark Department of Veterinary Disease Biology, University of Copenhagen, Denmark The Marine Laboratory, Aberdeen, Scotland National Institute of Aquatic Resources, Technical University of Denmark, Denmark Department of Veterinary Disease Biology, University of Copenhagen, Denmark National Veterinary Institute, Technical University of Kirsten Engell-Sørensen Fishlab, Denmark Norwegian College of Fishery Science , University of Department of Veterinary Disease Biology, University of Copenhagen, Denmark Dansk Akvakultur, Denmark National Institute of Aquatic Resources, Technical University of Denmark Department of Veterinary Disease Biology, University of Copenhagen, Denmark Akva Group, Denmark National Food Institute, Technical University of Denmark, Denmark East China Normal University, School of Life Science, Shanghai, China Aquatic Animal Health Section, Pacific Biological Station, Fisheries and Oceans Canada, Canada Department of Veterinary Disease Biology, University of Copenhagen, Denmark Department of Veterinary Disease Biology, University of Copenhagen, Denmark Department of Veterinary Disease Biology, University of Copenhagen, Denmark National Veterinary Institute, Technical University of Surname E-mail
Affilation
National Veterinary Institute, Technical University of Denmark National Institute of Aquatic Resources, Technical University of Denmark Department of Veterinary Disease Biology, University of Copenhagen, Denmark Skretting Aquaculture Research Centre, Stavanger, Norway Department of Veterinary Disease Biology, University of Copenhagen, Denmark Department of Veterinary Disease Biology, University of Copenhagen, Denmark National Food Institute, Technical University of Denmark, Denmark School of Aquaculture, University of Tasmania, Tasmania Intervet/Schering-Plough, Denmark National Veterinary Institute, Technical University of Denmark Department of Veterinary Disease Biology, University of Copenhagen, Denmark Department of medical Biology, University of Yaseelan Palarasah Southern Denmark, Denmark Department of Veterinary Disease Biology, University of Copenhagen, Denmark Department of medical Biology, University of Southern Denmark, Denmark National Veterinary Institute, Technical University of Denmark Department of Veterinary Disease Biology, University of Copenhagen, Denmark National Food Institute, Technical University of Denmark, Denmark Department of Aquaculture, Estonian University of Life Sciences, Estonia Department of medical Biology, University of Mikkel-Ole Skjødt Southern Denmark, Denmark Department of medical Biology, University of Southern Denmark, Denmark Department of Veterinary Disease Biology, University of Copenhagen, Denmark Department of Veterinary Disease Biology, University of Copenhagen, Denmark Department of Veterinary Disease Biology, University of Copenhagen, Denmark Equipe of Pathogen and Environment, University of Montpellier, France Department of medical Biology, University of Southern Denmark, Denmark Department of Veterinary Disease Biology, University of Copenhagen, Denmark

Source: http://www.dafinet.dk/Abstract_books_files/DAFINET%20November%202010.pdf