Immune gene expression in rainbow trout experimentally infected with simplex larvae

Haarder, Simon; Kania, Per Walter; Buchmann, Kurt

Publication date: 2012

Citation for published version (APA): Haarder, S., Kania, P. W., & Buchmann, K. (2012). Immune gene expression in rainbow trout experimentally infected with Anisakis simplex larvae. Abstract from DAFINET: Immune Responses in Fish, Frederiksberg, Denmark.

Download date: 27. Sep. 2021 DAFINET WORKSHOP

IMMUNE RESPONSES IN FISH November 6th and 7th , 2012

Venue: Organised by: Lecture Theatre 1-01 Danish Fish Immunology Research Bülowsvej 17 Centre and Network 1870 Frederiksberg www.dafinet.dk Denmark University of Copenhagen Frederiksberg Denmark Book of abstracts

DAFINET November 2012 University of Copenhagen DAFINET is supported by the Danish Council for Strategic Research

The book of abstracts is edited by Per W. Kania and Kurt Buchmann

Illustrations by Kurt Buchmann

Printed by Frederiksberg Bogtrykkeri 2012

Frederiksberg, Denmark

DAFINET WORKSHOP Danish Fish Immunology Research Centre and Network

IMMUNE RESPONSES IN FISH November 6 th and 7 th , 2012

Venue: Organised by: Lecture Theatre 1-01 Danish Fish Immunology Research Bülowsvej 17 Centre and Network 1870 Frederiksberg www.dafinet.dk Denmark University of Copenhagen Frederiksberg Denmark

Program Tuesday November 6 th , 2012

10.00 DAFINET Board meeting 10.00-12.00. Only for board members Scientific program 13.00 Welcome address by DAFINET leader Kurt Buchmann Laboratory of Aquatic Pathobiology, Department of Veterinary Disease Biology, University of Copenhagen, Denmark 13.15 Research director Scott LaPatra Clear Springs Foods, Inc., Research Division, Buhl, Idaho, USA Immunization of rainbow trout against flavobacteriosis 13:45 Professor Barbara Nowak National Centre for Marine Conservation and Resource Sustainability, AMC, University of Tasmania, Australia Yersiniosis vaccine research update 14.15 Post doc Jiwan Chettri Laboratory of Aquatic Pathobiology, Department of Veterinary Disease Biology, University of Copenhagen, Denmark Immune response of rainbow trout juveniles to the protozoan parasite Ichthyobodo necator : immunohistochemical and gene expression studies

14.45 Coffee break 15.30 Professor Barbara Nowak National Centre for Marine Conservation and Resource Sustainability, AMC, University of Tasmania, Australia Immune response of Southern Bluefin Tuna 16.00 M. Sc. student Simon Haarder Laboratory of Aquatic Pathobiology, Department of Veterinary Disease Biology, University of Copenhagen, Denmark Immune gene expression in rainbow trout experimentally infected with Anisakis simplex larvae 16.30 Senior research scientist Bertrand Collet Marine Laboratory, Ellis Building, Aberdeen, UK Development of a non-lethal sampling method to monitor immune response and disease progression in salmonid fish 17:00 Wrap up by DAFINET leader Kurt Buchmann 18.00 Dinner at Stigbøjlen 7, building 1-35, Frederiksberg Campus

Program Wednesday November 7 th , 2012

10.00 DAFINET leader Kurt Buchmann: Welcome to the second day of the workshop 10.10 Professor Barbara Nowak National Centre for Marine Conservation and Resource Sustainability, AMC, University of Tasmania, Australia – development of new management strategies 10.40 Ph.D. student Sidhartha Desmukh Laboratory of Aquatic Pathobiology, Department of Veterinary Disease Biology, University of Copenhagen, Denmark Characterization of central cellular and humoral mechanisms responsible for protection of vaccinated rainbow trout against Yersinia ruckeri by the use of histology and immunohistochemistry 11.00 Ph.D. student Bartolomeo Gorgoglione Scottish Fish Immunology Research Centre, School of Biological Sciences, University of Aberdeen, UK A strong antiviral response is elicited by viral hemorrhagic septicemia virus infection in brown trout 11.20 Ph.D. student Melanie Andrews Norwegian School of Veterinary Sciences, Oslo, Norway Wound closure and initial healing processes following adipose fin clipping of Atlantic 11.40 Post doc Maki Ohtani Laboratory of Aquatic Pathobiology, Department of Veterinary Disease Biology, University of Copenhagen, Denmark Antiviral activities of pattern recognition receptors, LGP2 and MDA5 in Japanese flounder, Paralicthys olivaceus

12.00 Lunch at Stigbøjlen 7, building 1-35

Program Wednesday November 7 th , 2012

13.00 Ph.D. student Maya Henriksen Division of Veterinary Diagnostics and Research, National Veterinary Institute, Technical University of Denmark The role of the gills as potential portal of entry in rainbow trout fry syndrome 13.20 Ellen Lorenzen Department of Poultry, Fish and Fur ; National Veterinary Institute, Technical of University Denmark Improved protection of rainbow trout against furunculosis by an autologous vaccine under experimental conditions 14.40 Post doc Milena Monte Scottish Fish Immunology Research Centre, School of Biological Sciences, University of Aberdeen, Aberdeen, UK Identification and expression analysis of four cytokine receptor family B (CRFB) members in rainbow trout ( Oncorhynchus mykiss ) 14.00 Coffee break 14.40 Ph.D. student Umaporn Khimmakthong Department of Molecular Biotechnology and Bioinformatics, Prince of Songkla University, Hat Yai, Thailand Immunization of pacific white shrimp by phagocytosis activating protein gene

15.00 Research assistant Rezkar Jafaar Laboratory of Aquatic Pathobiology, Department of Veterinary Disease Biology, University of Copenhagen, Denmark Gut microbiota changes in rainbow trout, Oncorhynchus mykiss (Walbaum) during organic acid feed supplementation and Yersinia ruckeri infection 15.20 Ph.D. student Anna Shönhertz Center for Quantitative Genetics and Genomics , Department of Molecular Biology and Genetics, Aarhus University, Denmark Inter-species transmission of viral haemorrhagic septicaemia virus between turbot (Scophthalmus maximus) and rainbow trout ( Oncorhynchus mykiss) 15.40 Final discussion and conclusions of the workshop

Abstracts IMMUNIZATION OF RAINBOW TROUT AGAINST FLAVOBACTERIOSIS

Scott E. LaPatra

Clear Springs Foods, Inc., Research Division, Buhl, Idaho 83316, USA

Flavobacteriosis is primarily caused by psychrophilum and F. columnare . Together they are the major bacterial pathogen contributors to losses in the rainbow trout ( Oncorhynchus mykiss Walbaum) farming industry in Idaho, USA and freshwater salmonid farming around the world. Outbreaks of Flavobacteriosis can result in significant mortality but further losses due to deformities and reduced growth in survivors contributes to additional and potentially greater economic impacts for the farmer.

Disease prevention is essential to the continued development of aquaculture around the world and remains a primary strategy for avoiding Flavobacteriosis, however vaccination is the most effective method of preventing or minimizing disease. Killed, attenuated, subunit and a variety of other vaccine formulations against Flavobacteriosis have been developed and tested over the years and despite a number of publications there are still no commercially available vaccine(s) for rainbow trout.

One slightly different, alternative “vaccine” we have evaluated is an Enterobacter sp. surrogate vaccine/immunostimulant against F. psychrophilum . Originally developed as a probiotic it shows promise, at least for short term protection when delivered by intramuscular injection. Equally important to the development of Flavobacteriosis vaccines is the development of mass immunization strategies. An efficacious vaccine is only of use to producers if it can be delivered cost effectively to small fish. With this in mind, a rifampicin attenuated F. psychrophilum vaccine has been developed and field tests suggest it can be effective when delivered by immersion. Similarly a live attenuated F. columnare vaccine licensed for use in channel catfish is also being evaluated for use in rainbow trout.

Presenting author: Scott E. LaPatra, [email protected]

YERSINIOSIS VACCINE RESEARCH UPDATE Barbara Nowak

National Centre for Marine Conservation and Resource Sustainability, AMC, University of Tasmania, Launceston, Tasmania, Australia

Yersiniosis is a bacterial disease affecting Atlantic salmon in hatchery and after transfer to the sea. While there are commercial vaccines available, outbreaks still occur due to the presence of carriers.

Using cDNA microarray we identified the expression of 6 genes in response to infection and 4 genes associated with the protective response to yersiniosis. Analysis by real-time PCR confirmed that three immunologically relevant genes, namely a cathelicidin (47-fold) and a C-type lectin (19-fold) increased in response to yersiniosis. Including collagenase (17-fold increase), an important tissue remodelling and repair enzyme, these genes represent 3 of 6 non-protective and/or pathological responses to yersiniosis. Genes associated with the protective host response included an immunoglobulin gene and a selenoprotein that showed significant fold changes (15-fold increases each), highlighting the importance of -mediated protection against yersiniosis. A transcriptional biosignature consisting of predominantly immune-relevant genes (14 up and 3 down- regulated) in the gills of Atlantic salmon after immersion vaccination and before bacterial challenge was identified. This biosignature may be used as a surrogate of protection and therefore as a predictor of vaccine success against yersiniosis.

We have tested an effect of different inactivation of Y. ruckeri on vaccine efficacy. Further research on oral delivery of the vaccine will be also discussed.

References Bridle AR, Koop BF, Nowak BF (2012) Identification of Surrogates of Protection against Yersiniosis in Immersion Vaccinated Atlantic Salmon. PLoS ONE 7(7): e40841. doi:10.1371/journal.pone.0040841 Presenting author: Barbara Nowak , [email protected]

IMMUNE RESPONSE OF RAINBOW TROUT JUVENILES TO THE PROTOZOAN PARASITE ICHTHYOBODO NECATOR : IMMUNOHISTOCHEMICAL AND GENE EXPRESSION STUDIES

1Chettri J.K., 1Kuhn J., 1Jaafar R.M., 1Kania P.W., 2Møller O.S. and 1 Buchmann K.

Laboratory of Aquatic Pathobiology, 1Department of Veterinary Disease Biology, University of Copenhagen, Denmark 2Zoological Museum, University of Copenhagen, Denmark

The present study was performed to examine the immune response mounted by rainbow trout juveniles against the protozoan parasite Ichthyobodo necator . An in vivo infection of the parasite was established in the laboratory by cohabitating infected fish with naive fish. An experimental infection was then initiated and the course of infection was followed. Samples were taken at regular time points from both uninfected and infected groups. Three fish from each group were examined for infection intensity at day 5, 9 and 14 post infection. In addition, samples were preserved for immunohistochemistry at day 5, 9 and 14 post infection and for qPCR at day 14 post infection.

Immunohistochemistry using a monoclonal antibody against trout IgM showed a significant increase in of IgM positive cells in the skin of the infected fish. The density of CD8 cells showed a declining pattern during the course of infection but did not vary significantly. No significant change in expression of IgT antibody was observed. The qPCR study supported the findings from the immunohistochemistry showing a significant increase in expression of IgM at the transcriptional level. Other immune factors such as lysozyme, SAA, cathelicidin 2 and cytokines (IL1 β, IL6, IL8, IL10, IL4/13a) showed a significant increase after infection. This is the first report on the immune response of rainbow trout towards I. necator infection.

Presenting Author: Jiwan Chettri, [email protected]

IMMUNE RESPONSE OF SOUTHERN BLUEFIN TUNA

Barbara Nowak

National Centre for Marine Conservation and Resource Sustainability, AMC, University of Tasmania, Launceston, Tasmania, Australia

Southern Bluefin Tuna ( Thynnus maccoyii ) is a commercially important species in Australia. While in general there are few health problems with blood flukes Cardicola spp. there are the highest risk for the performance of the ranched tuna. Humoral immune response, including C. forsteri specific antibody, lysozyme activity, and alternative complement activity, was investigated in ranched tuna.

Immune response developed concurrently with C. forsteri infection. We also identified mRNA sequence of five potential inflammatory mediators – TNF α (1 and 2), IL-1β, IL-8, and Cox2 – for southern bluefin tuna and showed that TNF α (2), IL-1β, IL-8, and Cox2 are induced following LPS stimulation of both peripheral blood leukocytes and head kidney homogenates. The transcription of cytokines TNF α, IL-1β and IL-8 following LPS stimulation was accelerated at 25°C. Peak induction reached comparable levels for each transcript at both 18°C and 25°C during the 24 h. Partial mRNA sequence was also identified for both the constitutively expressed and heat inducible chaperone proteins Hsc70 and Hsp70, and 24h incubation at 25°C induced Hsp70 transcription in leukocyte but not in head kidney cell populations. LPS stimulation failed to induce Hsc70 or Hsp70 at either incubation temperature; however, in 25°C LPS stimulated leukocytes, rapid induction of cytokines was followed by the heat-induced transcription of Hsp70. Transcriptional expression of Hsp70 and IL-8 were correlated following LPS/heat shock stimulation of leukocytes.

References Kirchhoff NT, Leef MJ, Valdenegro V, Hayward CJ, Nowak BF (2012) Correlation of Humoral Immune Response in Southern Bluefin Tuna, T. maccoyii , with Infection Stage of the Blood Fluke, Cardicola forsteri . PLoS ONE 7(9): e45742. doi:10.1371/journal.pone.0045742

Polinski M, Bridle AR, Nowak BF (submitted) Temperature-induced transcription of inflammatory mediators and the influence of Hsp70 following LPS stimulation of southern bluefin tuna peripheral blood leukocytes and kidney homogenates

Presenting author: Barbara Nowak , [email protected]

IMMUNE GENE EXPRESSION IN RAINBOW TROUT EXPERIMENTALLY INFECTED WITH ANISAKIS SIMPLEX LARVAE

Haarder S., Kania P.W., Bahlool Q.Z.M. and Buchmann K. Laboratory of Aquatic Pathobiology, Department of Veterinary Disease Biology, University of Copenhagen, Denmark

Basic immune response mechanisms in vertebrates against infections are still poorly understood. Fish hosts and fish parasitic may prove to be valuable laboratory models for elucidation of this question. In this study we have orally challenged rainbow trout ( Oncorhynchus mykiss ) with larvae of Anisakis simplex (Nematoda: ) and subsequently investigated the expression of 18 immune relevant genes. Spleens and livers weresampled at 1, 4 and 8 days post infection (d.p.i.) and processed in accordance with standard quantitative real-time PCR (qPCR) protocols. Gene expression data were analyzed with regard to the infection status of the challenged salmon at the time of necropsy; “worms rejected” or “worms present”. Experimental fish which had rejected the experimentally introduced worms were found to significantly up-regulate CD4 (6.06 fold change, 8 d.p.i.) in liver when compared to control fish. Genes encoding CD8 and immunoglobulin IgM were found to be significantly down-regulated 24 hours and 8 days post challenge in livers of infected fish, respectively. Complement factor C3 and precerebellin genes were, on the contrary, significantly up-regulated two-fold in liver samples from infected fish 4 days post infection. Significant up-regulation of SAA was observed in both groups and both tissues at primarily 1 and 4 days after parasite invasion. The findings presented will be discussed in the light of previously presented hypotheses on vertebrate immune reactions against parasitic invasions and it is suggested that studies based on fish-nematode infection models should be conducted for further elucidation of basic parasite-induced host reactions.

Presenting Author: Simon Haarder , [email protected]

DEVELOPMENT OF A NON -LETHAL SAMPLING METHOD TO MONITOR IMMUNE RESPONSE AND DISEASE PROGRESSION IN SALMONID FISH 1 Collet B., 2Secombes C.J., Monte 2M., 1Urquhart K., 3Adams A., 3Thompson K.

1Marine laboratory, Scotland, UK 2University of Aberdeen, UK 3University of Stirling, UK

Every year approx. 200,000 fish are used for fundamental or applied research in the UK. Traditional experimental infections require that animals are killed at regular intervals to measure the progression of pathology and/or immune response. In addition to requiring a high number of animals this practice suffers from high inter-individual variability.

Marine Scotland, the Universities of Aberdeen and Stirling were awarded a 2 year research project by the National Centre for the Replacement, Refinement and Reduction of in science (NC3Rs) aiming at developing a non-lethal experimental infection model in order to i) reduce the number of fish required in experimentation, ii) refine the scientific output from infectivity studies and iii) replace the use of animals by the development of in vitro tests correlated with parameters traditionally measured in vivo. Viral or bacterial pathogens of Atlantic salmon ( Salmo salar ) and rainbow trout ( Oncorhynchus mykiss ) that cause serious economical loss to the UK fish farming industry will be used in successive experimental infections. A “low stress” method will be utilised to collect repeatedly small amounts of blood from the same individual. The establishment of correlations between immune and welfare parameters, viraemia/bacteraemia and pathological status will potentially allow prediction of the disease resistance status of livestock, virulence of pathogens and efficacy of vaccines or immunostimulants. In addition to the contribution to the 3Rs principle, this project has the potential to improve the quality of scientific output in fish disease research.

Presenting author: Bertrand Collet , [email protected]

AMOEBIC GILL DISEASE – DEVELOPMENT OF NEW MANAGEMENT STRATEGIES Barbara Nowak

National Centre for Marine Conservation and Resource Sustainability, AMC, University of Tasmania, Launceston, Tasmania, Australia

Amoebic Gill Disease (AGD) is the most serious health problem for Atlantic salmon farmed in Tasmania, contributing up to 20% to production costs. It is now becoming an issue in other salmon producing countries, including Scotland and Ireland. The only commercially available treatment is freshwater bathing, which has to be repeated on regular basis, more frequently when water temperature is higher. We have been developing management strategies including vaccination, immunostimulation and treatment with hydrogen peroxide. In a preliminary study we assessed the systemic and mucosal immune responses of Atlantic salmon exposed to standard antigens (DNP and FITC) administered by different techniques as a means to identify the most appropriate delivery method for prospective vaccines for AGD.

We are currently testing experimental vaccines against AGD. Experimental oral delivery of immunostimulants showed promising results reducing mortalities, however neither lysozyme nor complement activity were changed. In vitro and in vivo experiments with hydrogen peroxide suggested that it could be used as an alternative treatment to freshwater bathing.

References Adams M, Crosbie P, Nowak B (in press) Preliminary success using hydrogen peroxide to treat Atlantic salmon Salmo salar L. affected with experimentally induced Amoebic Gill Disease (AGD). Journal of Fish Diseases. Valdenegro-Vega VA, Crosbie P, Vincent B, Cain KD, Nowak BF (submitted) Effect of immunization route on mucosal and systemic immune response in Atlantic salmon (Salmo salar ) Presenting author: Barbara Nowak , [email protected]

CHARACTERIZATION OF CENTRAL CELLULAR AND HUMORAL MECHANISMS RESPONSIBLE FOR PROTECTION OF VACCINATED RAINBOW TROUT AGAINST YERSINIA RUCKERI INFECTION BY THE USE OF HISTOLOGY AND IMMUNOHISTOCHEMISTRY

Deshmukh S., Chettri J.K. and Buchmann K. Laboratory of Aquatic Pathobiology, Department of Veterinary Disease Biology, University of Copenhagen, Denmark

We investigated, by the use of histology and immunohistochemistry, the changes of tissue architecture and transformation of various resident cell types in immune organs of vaccinated fish responding to Yersinia ruckeri serotype 01, biotype 2 infection. In addition, by a similar approach we characterized the cellular basis for differential efficacy of two commercial vaccines against ERM. Rainbow trout were challenged by intra-peritoneal injection with Yersinia ruckeri serotype 01, biotype 2 and the patho-morphological alterations were recorded during early ( i.e. 3 and 7 day post infection) and late course of infection (21 and 30 days post infection at 4 month post- vaccination and at day 14 post-infection in case of 8 month post vaccination). Comparatively delayed and lesser degree of tissue damage and early restoration to normal histoarchitecture of both immune was noticed in fish vaccinated with AquaVac® RELERA™ compared to AquaVac® ERM. The differential occurrence of CD8 α and IgM positivity characterized the first decisive processes in vaccinated rainbow trout. These cell mediated and humoral immune mechanisms occurred significantly faster and more pronounced in AquaVac® RELERA™ vaccinated fish when compared fish vaccinated with AquaVac® ERM and unvaccinated fish.

Presenting author: Sidhartha Desmukh, [email protected]

A STRONG ANTIVIRAL RESPONSE IS ELICITED BY VIRAL HEMORRHAGIC SEPTICEMIA VIRUS INFECTION IN BROWN TROUT 1,2Gorgoglione B., 1Zou J., 2Taylor N. and 1Secombes C.J. 1Scottish Fish Immunology Research Centre, School of Biological Sciences, University of Aberdeen, Aberdeen, UK 2CEFAS, Weymouth Laboratory, Weymouth, Dorset, UK

Viral hemorrhagic septicemia (VHS) is one of the most economically important diseases of wild and cultured fish world-wide, resulting in mortalities >80% in marine and freshwater susceptible species. Caused by an enveloped RNA virus (VHSV), (Fam. Rhabdoviridae ), it is a notifiable listed disease for OIE and European legislation (Council Directive 2006/88/EC). No practicable treatments are available, although DNA vaccination is giving encouraging experimental results but is still a long way from a commercial product.

Recently, many immune related genes have been identified in species, from interrogation of sequenced genomes and growing EST databases. Several studies have already focused on the characterisation of the host immune response mounted against VHSV, like the interferon (IFN) mediated response. To date, 2 groups of Type I IFNs, consisting of either 2 or 4 cysteines, have been characterised in fish, with a further division into 6 phylogenetic subgroups. Like their counterparts in higher vertebrates, they are activated at the early stage of viral infection and play a vital role in co-ordinating innate antiviral defence.

RT-qPCR gene screening was carried out on laboratory-reared brown trout ( Salmo trutta ) to characterise the type of immune response elicited within the kidney parenchyma, after an in vivo bath challenge using the first UK VHSV strain isolated from salmonids (sub-lineage VHSV Ia).

Brown trout showed a significantly delayed (1st episode after 8 p.i.) and reduced cumulative mortality (<6%) when compared with rainbow trout (Oncorhynchus mykiss ). In the early stages of VHS infection, a very strong induction of IFNs, IFN-induced transcripts and pro-inflammatory genes was seen and this was highly correlated with virus systemic replication (assessed by TCID50/g and by RT-qPCR assessment of VHSV gpG). The gene expression results in brown trout confirm and provide more clues on the immune response elicited by this viral infection in a relatively more resistant trout species.

Presenting author: Gorgoglione Bartolomeo, [email protected]

WOUND CLOSURE AND INITIAL HEALING PROCESSES FOLLOWING ADIPOSE FIN CLIPPING OF ATLANTIC SALMON Andrews M. and Midtlyng P.J. Norwegian School of Veterinary Sciences, Oslo, Norway Adipose fin clipping is a commonly employed marking technique used in both research and within the aquaculture industry, yet few studies have been conducted on the initial wound closure post- clipping. Having open wounds for an extended time may have a significant effect on overall fish health and welfare and may have an impact on experimental results if used as a batch marking method. An experiment was conducted to describe the wound closure processes over a 72h period post-clipping and whether water temperature had an effect on the rate of wound closure. A total of 204 Atlantic salmon (mean 36.1g; range 30.7 – 43.8g) were randomly placed in 3 groups (n = 66) each kept at a different water temperature (4, 10 and 14°C). Six fish were sampled from each tank at 11 time points post-clipping (2, 4, 6, 12, 18, 24, 30, 36, 48, 60 and 72h) with an additional 6 fish being sampled at 0h. Samples were then prepared using histological methods and examined to determine and describe the wound closure processes over a period of 72 h post-clipping. In addition the effects of differing water temperatures on the rate of wound closure and healing.

The results from this experiment may assist in determining whether adipose fin clipping is a viable marking technique for mass marking and hopefully has a relatively low impact on overall fish health and welfare. The results will be discussed in this presentation.

Presenting author: Melanie Andrews, [email protected]

ANTIVIRAL ACTIVITIES OF PATTERN RECOGNITION RECEPTORS , LGP2 AND MDA5 IN JAPANESE FLOUNDER , PARALICTHYS OLIVACEUS

1, 2Ohtani M. 1, 2, 2Hikima J., 2Jung T.S. and 2, 3Aoki T.

1Laboratory of Aquatic Pathobiology, Department of Veterinary Disease Biology, University of Copenhagen, Denmark

2Aquatic Biotechnology Center, College of Veterinary Medicine, Gyeongsang National University, South Korea 3Consolidated Research Institute for Advanced Science and Medical Care, Waseda University,

Retinoic acid-inducible gene-I like receptors (RLRs) family is one of pattern recognition receptors (PRRs), and is very important for intercellular immune response to recognized the viral RNAs. MDA5 (melanoma differentiation-associated gene 5) and LGP2 (laboratory genetics and physiology 2) belong in RLR family. These molecules enhance gene expression of type-I interferon (IFN-I) and inflammatory cytokines.

The expression of MDA5 and LGP2 mRNA were strongly up-regulated by poly I:C stimulation or VHSV infection in vivo and in vitro . To reveal the antiviral activity of the MDA5 and LGP2, expression vectors were constructed and used for antiviral assay against VHSV, HIRRV and IPNV in vitro. Over-expression of MDA5 and LGP2 tightly repressed the viral replication which was attributed to the up-regulation of interferon inducible genes (Mx and ISG15). These results indicated that MDA5 and LGP2 can induce the inhibition of viral replication through the induction of interferon inducible gene expression. To confirm the dsRNA receptors induce the IFN-I gene expression, both receptors were co-transfected with IFN-I promoter. The IFN-I promoter activity showed that LGP2 and MDA5 enhanced IFN-I promoter activity with intracellular poly I:C stimulation. These results suggested that Japanese flounder MDA5 and LGP2 recognize cytosolic dsRNA and induce the production of type I IFN for antiviral response.

Presenting author: Maki Ohtani, [email protected]

THE ROLE OF THE GILLS AS POTENTIAL PORTAL OF ENTRY IN RAINBOW TROUT FRY SYNDROME

1Henriksen M.M.M, 2Kania P.W., 1Madsen L., 2Buchmann K. and 1Dalsgaard I.

1National Veterinary Institute, Technical University of Denmark 2laboratory of Aquatic Pathobiology, Department of Veterinary Disease Biology, University of Copenhagen, Denmark

The Gram negative fish pathogen Flavobacterium psychrophilum is the causative agent of rainbow trout fry syndrome (RTFS), also known as BCWD (bacterial cold water disease). While injection- based experimental challenges with F. psychrophilum have been standardized and result in high mortality rates, the same has not been the case for immersion based models. However, injection is not a proper approach for investigations of the immune response since the first line of defense is bypassed. This study aims at understanding the immune response in the gills following infection as well as their possible role as portal of entry.

A bath model, using hydrogen peroxide (H 2O2) as a pre-treatment stressor to increase the number of infected fish, was used for obtaining samples. The experimental setup consisted of four treatment groups; 1) untreated controls, 2) H 2O2, 3) F. psychrophilum and 4) H 2O2 and F. psychrophilum . The use of this model also provides information regarding the impact of H 2O2 alone, though this was not the main objective. Samples were taken 4 hours, 2 days, 5 days and 8 days after infection and preserved for later processing. Two methods are employed in this study. Firstly, quantitative real- time PCR will be used to investigate the immune response in the gills after immersion exposure to F. psychrophilum . Secondly, fluorescent in situ hybridization will be used to visualize pathogen entry and spread in gills and head region.

The results will be presented at the workshop.

Presenting author: Maya Henriksen , [email protected]

IMPROVED PROTECTION OF RAINBOW TROUT AGAINST FURUNCULOSIS BY AN AUTOLOGOUS VACCINE UNDER EXPERIMENTAL CONDITIONS

1Lorenzen E., 1 Kjær T.E., 2Henriksen N.H., 1Dalsgaard I., 1,3 Holten-Andersen L., 4Madsen S.B., 5Krossøy B., 3Buchmann K. and 1Lorenzen N.

1National Veterinary Institute, DTU, Denmark 2Danish Aquaculture, Silkeborg, Denmark 3Laboratory of Aquatic Pathobiology, University of Copenhagen, Denmark 4S. B. Madsen Veterinary Services, Denmark 5Vaxxinova, Cuxhafen,

Despite vaccination with oil-adjuvanted vaccines against vibriosis and furunculosis, sea reared rainbow trout in Denmark often encounter outbreaks of furunculosis during warm summer periods. To address this issue under experimental conditions, two groups of rainbow trout were vaccinated by i.p. injection with two different oil-adjuvanted vaccines: (1) a commercial vaccine comprising Vibrio anguillarum serotype O1 and O2, and subspecies salmonicida bacterins, with all bacteria originating from Atlantic salmon , and (2) an experimental vaccine based on cultures of the same bacterial species originating from rainbow trout reared in Danish sea farms. The experiment also included a third group of non-vaccinated controls. All fish were individually chip-tagged to allow mixing of all groups in three replicate aquaria. After 770 dg (day degrees) or 77 days at 10 ⁰C, half of the fish in each group were challenged by i.p. injection of 1x105 cells of the A. salmonicida isolate used in the experimental vaccine. The other half was tagged by cutting off the adipose fin (non-injected cohabitants) .

While the non-vaccinated, i.p.-injected fish all died within 2 weeks, a certain level of protection was evident among the vaccinated groups although high mortality also occurred here. No mortality/clinical disease was evident among the non-injected cohabitants. However, when the water temperature was gradually risen to 15-17-20 ⁰C, the cohabitants started to die. Some variability was evident between replicate tanks, but the experimental vaccine tended to provide better protection than the commercial counterpart. The results indicate that tailor-making of a vaccine against furunculosis for sea reared rainbow trout in DK is an important approach for optimal protection.

Presenting author: Ellen Lorenzen, [email protected]

IDENTIFICATION AND EXPRESSION ANALYSIS OF FOUR CYTOKINE RECEPTOR FAMILY B (CRFB) MEMBERS IN RAINBOW TROUT (O NCORHYNCHUS MYKISS Monte M.M., Wang T., Costa M.M., Secombes C.J.

Scottish Fish Immunology Research Centre, School of Biological Sciences, University of Aberdeen, Aberdeen, UK. The interleukin (IL)-10 cytokine family, which includes cytokines such as IL-10, IL-20 and IL-22, are highly pleiotropic and mediate a variety of activities, including immune suppression and antibacterial immunity. To exert their functions they signal through a heterodimeric receptor composed of a subunit with a long intracellular domain (type R1) and a subunit with a short intracellular domain (type R2 type). In this study we report the identification of three R1 type receptors (named CRFB7, CRFB8a and CRFB8b) and one R2 type receptor (named CRFB4) in rainbow trout. The nomenclature of the receptors was supported by homology analysis, conserved motifs and phylogenetic tree analysis, confirming they belong to the piscine CRFB family. For instance, they all displayed the presence of conserved fibronectin type-III domains.

Expression analysis in tissues collected from healthy fish revealed different patterns of expression for each receptor, suggesting their potential involvement in different responses. When studying the modulation of all genes in cell lines and primary cultures, a greater effect was observed in cell lines with the expression of most receptors being affected after incubation with microbial mimics (LPS and PolyI:C) or the pro-inflammatory cytokine rIFN-γ. This suggests their potential role in inflammatory responses and microbial defence.

Presenting author: Milena Monte, [email protected]

IMMUNIZATION OF PACIFIC WHITE SHRIMP BY PHAGOCYTOSIS ACTIVATING PROTEIN GENE

1Khimmakthong U., 1,2 Deachamag P. and 1,2 Chotigeat W.

1Department of Molecular Biotechnology and Bioinformatics, Faculty of Science, Prince of Songkla University, Hat Yai, Thailand

2Center for Genomics and Bioinformatics Research

The phagocytosis activating protein ( PAP ) gene has been isolated from Penaeus monodon infected with the virus (WSSV). The PAP has capacity to induce phagocytosis of shrimp hemocytes. Previous studies showed that the percentage of phagocytosing hemocytes increased when the hemocytes were incubated with the recombinant protein glutathione-S- transferase-PAP (GST-PAP). Moreover, shrimps injected intramuscularly with GST–PAP protein showed increasing survival rate of the shrimp after being infected with WSSV.

In this study, PAP gene was cloned into the eukaryotic expression vector phMGFP . Shrimps (Litopenaeus vannamei ) were each injected with 40 µg of PAP-phMGFP . The analysis of immune response at 3, 7 and 10 days after immunization revealed that the immunized shrimp showed the highest expression of PAP and prophenoloxidase ( proPO ) gene at 7 days after immunization. The survival rate of shrimp was 86.61% after challenge with WSSV. In addition, quantitative analysis of WSSV in shrimp showed that the copies of WSSV were significantly reduced in the PAP-phMGFP injected shrimp. This finding can conclude that PAP-phMGFP can increase the immune response in shrimp and can protect shrimp against WSSV.

Presenting author: Umaporn Khimmakthong, [email protected]

GUT MICROBIOTA CHANGES IN RAINBOW TROUT , ONCORHYNCHUS MYKISS (W ALBAUM ) DURING ORGANIC ACID FEED SUPPLEMENTATION AND YERSINIA RUCKERI INFECTION 1Jaafar R.M., 1Kania P.W., 2Larsen A.H., 3Nielsen D.S., 4Fouz B., 5Browdy C. and 1Buchmann

1Laboratory of Aquatic Pathobiology, Department of Veterinary Disease Biology, University of Copenhagen, Denmark 2BioMar A/S, Denmark, 3Department of Food Science, University of Copenhagen, Denmark, 4Department of Microbiology and Ecology, University of Valencia, Burjassot, Valencia, , 5Novus International, St. Charles, MO, USA Few studies investigating the role of gut microbiota (GM) in health and disease of fish have been conducted. This basic parameter may none-the-less be of importance also to fish health because intestinal bacteria contribute to digestion of food, provision of essential nutrients, gut development, gut maturation, regulation of the immune system and may thereby prevent pathogen colonization. It is therefore relevant to investigate the association between GM and health also in fish. We have studied how the use of formic and propionic acid (product name Mera™ Cid) (alone or in combination with immunostimulants) as inclusion in aquaculture feeds affects the autochtonous gut microbiota in fish. Further the effect on GM of Yersinia ruckeri exposure was evaluated. For monitoring basic health parameters of fish during experimentation haemoglobin and lysozyme were measured.

DGGE analysis of bacterial community profiles revealed that fish fed Mera™ Cid for 14 and 45 days obtained a significantly different intestinal microbiota compared to non-Mera™ Cid fed fish. These variations showed no association with increased plasma lysozyme activity as this was only seen at day 45 in infected control fish but it was noted that Mera™ Cid fed fish also showed haemoglobin increases at day 14. Further, Yersinia ruckeri infection was found to influence the rainbow trout GM which suggests that there exists a link in trout between GM and health. Future studies should elucidate the responsible mechanisms – which may include interactions between microorganisms and/or interactions between host responses and microorganisms.

Presenting Author: Rezkar Jafaar , [email protected]

INTER -SPECIES TRANSMISSION OF VIRAL HAEMORRHAGIC SEPTICAEMIA VIRUS BETWEEN TURBOT (SCOPHTHALMUS MAXIMUS ) AND RAINBOW TROUT (ONCHORHYNCHUS MYKISS )

1Schönherz A.A., 2Lorenzen N., 2Einer-Jensen K. 1Department of Molecular Biology and Genetics, Aarhus University, Denmark 2Department of Poultry, Fish and Fur Animals; National Veterinary Institute, Technical University of Denmark

Viral haemorrhagic septicaemia is a serious viral disease of teleost fish with high economic impact on the aquaculture industry. The disease is caused by the viral haemorrhagic septicaemia virus (VHSV), an RNA virus belonging to the family Rhabdoviridae. Compared to other rhabdoviruses infecting fish, VHSV has an exceptional wide host range of more than 70 species across marine and aquatic environments. To establish such a wide host range host-specific adaptation would be disadvantageous, nevertheless, host-specific differences in pathogenicity have been observed for VHSV. The divergence in pathogenicity, however, is not fully resembled in the phylogeny, which indicates a correlation between geographic regions rather than host species. The objective of this study was to identify whether VHSV has the ability to transmit between different host species or whether viral transmission is restricedt to one host species through host-specific adaptation. To investigate the existence of inter-species transmission and host-specificity a cohabitation challenge between turbot and rainbow trout was conducted with turbot as donor- and rainbow trout as recipient host species. Turbot were ip challenged with a turbot- or a rainbow trout adapted VHSV isolate and subsequently grouped with naïve rainbow trout. Mortality and viral shed was monitored daily. Both virus isolates showed signs of host-specific adaptation based on differences in replication dynamics, viral production, and virulence. Host-specific adaptation, however, did not result in total restriction of inter-species transmission. Despite of host-specific adaptation, the rainbow trout adapted VHSV isolate was able to cause disease in turbot resulting in subsequent infection of cohabiting rainbow trout, thus indicating the existence of inter-species transmission of VHSV between turbot and rainbow trout.

Presenting author: Anna Schönherz, [email protected]

List of participants

First name Surname e-mail Affiliation Norwegian School of Veterinary Sciences,

Zaniar Abbas [email protected] Norway Department of Veterinary Disease Biology,

Azmi Al-Jubury [email protected] University of Copenhagen, Denmark Faculty of Science, University of Copenhagen,

Jørn Andreassen [email protected] Denmark Norwegian School of Veterinary Sciences,

Melanie Andrew [email protected] Norway Department of Veterinary Disease Biology,

Qusay Bahlool [email protected] University of Copenhagen, Denmark National Veterinary Institute,

Simona Bartkova [email protected] Technical University of Denmark Department of Veterinary Disease Biology,

Kurt Buchmann [email protected] University of Copenhagen, Denmark Department of Veterinary Disease Biology,

Jiwan Kumar Chettri [email protected] University of Copenhagen, Denmark Marine Laboratory, Ellis Building, Aberdeen, Bertrand Collet [email protected] UK National Veterinary Institute,

Inger Dalsgaard [email protected] Technical University of Denmark Department of Veterinary Disease Biology,

Sidhartha Deshmukh [email protected] University of Copenhagen, Denmark Scottish Fish Immunology Research Centre

Bartolomeo Gorgoglione [email protected] University of Aberdeen UK, UK Brian Department of Veterinary Disease Biology,

Hansen [email protected] Klitgaard University of Copenhagen, Denmark Department of Veterinary Disease Biology,

Iselin Jahre Hegna [email protected] University of Copenhagen, Denmark Rasmus Department of Veterinary Disease Biology,

Heinecke [email protected] Demuth University of Copenhagen, Denmark National Veterinary Institute,

Maya Henriksen [email protected] Technical University of Denmark Department of Veterinary Disease Biology,

Lars Holten-Andersen [email protected] University of Copenhagen, Denmark Department of Veterinary Disease Biology,

Simon Haarder [email protected] University of Copenhagen, Denmark Hans- National Veterinary Institute,

Ingerslev [email protected] Christian Technical University of Denmark Department of Veterinary Disease Biology,

Hannah Jensen [email protected] University of Copenhagen, Denmark Department of Veterinary Disease Biology,

Per Kania [email protected] University of Copenhagen, Denmark Department of Molecular Biotechnology and

Umaporn Khimmakthong [email protected] Bioinformatics, Prince of Songkla University, Hat Yai, Thailand

Scott LaPattra [email protected] Clear Springs Foods, Inc., Idaho, USA

Katherine Lester [email protected] Marine Laboratory, Aberdeen, UK

List of participants, continued

First name Surname e-mail Affiliation National Veterinary Institute,

Ellen Lorenzen [email protected] Technical University of Denmark National Veterinary Institute,

Lone Madsen [email protected] Technical University of Denmark Department of Veterinary Medical Science Alma

Luciana Mandrioli [email protected] Mater Studiorum Bologna, Department of Veterinary Disease Biology,

Sanaz Mazaheri [email protected] University of Copenhagen, Denmark Department of Veterinary Disease Biology,

Foojan Mehrdana [email protected] University of Copenhagen, Denmark Department of Veterinary Disease Biology,

Rzgar Jaarfar Mohammad [email protected] University of Copenhagen, Denmark Zoological Museum, Ole Sten Møller [email protected] University of Copenhagen, Denmark Scottish Fish Immunology Research Centre

Milena Monte [email protected] University of Aberdeen UK, UK Department of Veterinary Disease Biology, Lukas Neumann [email protected] University of Copenhagen, Denmark NCMCRS AMC,

Barbara Nowak [email protected] University of Tasmania, Tasmania

Jørgen Nylen [email protected] Intervet/Schering-Plough, Denmark Department of Veterinary Disease Biology,

Moonika Marana Olsen [email protected] University of Copenhagen, Denmark Department of Veterinary Disease Biology,

Maki Otani [email protected] University of Copenhagen, Denmark

Matthew Owen [email protected] Skretting, Norway Department of Veterinary Disease Biology,

Kristian Plet-Hansen [email protected] University of Copenhagen, Denmark National Veterinary Institute,

Jesper Skou Rasmussen [email protected] Technical University of Denmark Rømer Department of Veterinary Disease Biology,

Kasper [email protected] Villumsen University of Copenhagen, Denmark Department of Molecular Biology and Genetics, Anna Amanda Schönherz [email protected] Aarhus University, Denmark Department of Veterinary Disease Biology,

Diana Sindberg [email protected] University of Copenhagen, Denmark Department of Veterinary Disease Biology,

Jakob Skov [email protected] University of Copenhagen, Denmark Department of Veterinary Disease Biology,

Alf Skovgaard [email protected] University of Copenhagen, Denmark Section for Population Ecology and Genetics,

Sune Riis Sørensen [email protected] Technical University of Denmark Marine Biological Section,

Demeng Tan [email protected] University of Copenhagen

Illustrations by Kurt Buchmann