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Studies on Resistance to Infectious Pancreatic Necrosis Virus In Studies on Resistance to Infectious Pancreatic Necrosis Virus in Atlantic salmon (<Salmo salar) Karen Patricia Plant B.Sc. (Hons) Biology, M.Sc. Applied Fish Biology Thesis submitted to the University of Stirling for the Degree of Doctor of Philosophy ProQuest Number: 13916336 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a com plete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. uest ProQuest 13916336 Published by ProQuest LLC(2019). Copyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States C ode Microform Edition © ProQuest LLC. ProQuest LLC. 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 4 8 1 0 6 - 1346 Declaration I declare that this thesis has been compiled by myself and is the result of my own investigations. It has not been submitted for any other degree and all sources of information have been duly acknowledged. Karen Plant : T ' "v.'V' \ ■ i y j / i p ' r j I: ■" i > ‘ : \ j ? , 4 ^ 1 ::i:y :Uv ... y v ; (C -.1 , ' ,* ' ,'s ; " ;'c v ;; s ■: Abstract Infectious pancreatic necrosis virus (IPNV) is responsible for serious mortalities in post- smolt Atlantic salmon (Salmo salar) annually in both Scotland and Norway. The huge financial losses involved and the lack of control measures have prompted attempts to produce IPN vaccines and disease resistant strains of fish. A characteristic of IPNV is its ability to persist in a latent carrier state and to ensure stocks of fish are truly virus free there is a need for highly sensitive diagnostic methods. In this study microsatellites were employed to assign fish susceptible to IPN in the field to families. Laboratory challenges were performed on 24 families and various humoral immune parameters were measured. A variety of diagnostic techniques were also developed, their sensitivity and specificity evaluated and compared to tissue culture. Parental Atlantic salmon and offspring susceptible to IPN in the field were genotyped using a published multiplex microsatellite PCR adapted for use with fluorescent genotyping systems. The reliability and utility of the genotyping system was evaluated with positive results. The majority of susceptible fish could not be assigned to parents since the fish were found to be from a mixed stock. Various experimental challenges with IPNV infected tissue homogenate were performed on post-smolts, however they did not produce mortality or induce clinical IPN. As a result of this virus titre was used as an indirect measurement of disease resistance. Atlantic salmon post-smolts were found to effectively clear cell cultured IPNV, isolate Br from Shetland. A second Shetland isolate (Os) was found to actively replicate in the fish while isolate Br was rapidly cleared. To increase virulence isolate Os was passaged once further through fish prior to use in challenging 24 families of PIT tagged Atlantic salmon. At week 3 mean viral titres for each family ranged from 10 0 8 to 1011 TCID 5o/g kidney. Using these data six families were targeted to follow through to week 7, two with low titres, two with medium titres and two with high titres of IPNV. 0 8 15 At week 7 virus titres had decreased to between 10 ' and 1 0 '' TCIDso/g kidney, many of the fish in the families with low IPNV titres at week 3 had cleared the virus. Lysozyme and neutralising antibody responses were measured, however little or no correlation was found between either parameter and IPNV titre at weeks 3 or 7. The control fish were found to be positive for IPNV despite earlier negative testing. Reasons for this were unclear, the fish may have been exposed to the virus through the seawater inflow or alternatively the fish may have been carriers of the virus. Diagnostic techniques employed to detect IPNV were RT-PCR, ELISA and immunohistochemistry; cell culture was used as a comparison to the RT-PCR and ELISA. The RT-PCR was developed using two published IPNV primer sets both of which amplified regions on VP2, they were found to be specific to IPNV and together amplified 9 out of 10 serotypes of IPNV. The most sensitive primer set detected between 10 7 and 108 TCID 50 IPNV/ml in spiked tissue, in comparison to the ELISA which detected 1012'5 TCID 50 IPNV/ml in tissue homogenate. The AS-1 monoclonal antibody was used successfully in immunohistochemistry to identify IPNV in fixed tissue. The antibody was found to identify IPNV in pancreatic lesions from infected fish, but did not detect the virus in the gut. All of these methods were found to be less sensitive than cell culture, however they are very rapid and ELISA and RT-PCR may be used to confirm IPNV infection once CPE has been visualised in cell culture. Acknowledgements I would like to express my gratitude to the following people who have provided me with help and advice that has contributed to the completion of this thesis. Firstly, I would like to thank my supervisors Professor Brendan McAndrew and Dr. Alexandra Adams for their advice, support and unfaltering belief in me. I would also like to thank the technicians at the Institute, especially Gillian, Debbie, Maureen, Hilary, Linton, Jacquie and most of all Fiona for all her virology training. Many thanks to Dr. John Taggart for his help with family assignment, also to Dr. Margaret Cairney for advice on working with RNA. I would like to give special thanks to Professor Hugh Ferguson for valuable and much appreciated help with IPN pathology and immunohistochemistry. My thanks also go to the staff at Skerries Salmon for their assistance with mortality collection and to the islanders who made me feel at home, particularly Alice and Gibbie. I also want to thank Dr. Peter Dixon and Keith Way for help and advice since and during my time at the CEFAS laboratory and also for supplying IPNV serotypes. I would like to thank Dr. Ian Bricknell and the SERAD laboratory for providing tanks and for his advice on IPNV and Dr. David Smail for the Shetland IPNV isolate. Many thanks to Philip, Sally and Fiona at the SERAD Marine Laboratory at Aultbea for looking after my fish and for help with the horrible tasks of scanning fish and removing PIT tags. I would especially like to thank Sally and Magnus for their hospitality and refreshing view on life. I wish to thank my friends Sarah, Tara and Rachel for their support throughout the last three years. I cannot forget my friends within the Institute who have supported and listened to me, particularly Bryony, Carlos, Wendy, Polly, Mark, Steve, Fiona, Tom and Phoebe. Most of all I would like to thank Ruth for being there and for sharing many good times and many more bottles of wine. I especially want to thank my parents for all their love and support. iv Abbreviations M-l microlitre \lM micromolar ANOVA analysis of variance APS ammonium persulphate AS Atlantic salmon cells BF-2 bluegill fibroblast cells Br Brindister IPNV isolate BSA bovine serum albumin BSNV Blotched Snakehead Virus CEFAS Centre for the Environment Fisheries and Aquaculture CHSE-214 Chinook salmon embryo cells CPE cytopathic effect DEPC diethyl pyrocarbonate DNA deoxyribonucleic acid dNTP deoxynucleotide triphosphate dsRNA double stranded RNA EDTA ethylenediaminetetraacetic acid ELISA enzyme-linked immunosorbent assay EMEM Eagles minimum essential medium FBS foetal bovine serum fg fantogram FSG fish skin gelatin H&E haematoxylin and eosin HBSS Hank’s balance salt solution HEWL hen egg white lysozyme HRP horseradish peroxidase HW Hardy-Weinberg IBDV Infectious bursal disease virus DFAT immunofluorescence test IFN interferon IgG Immunoglobulin G IHC immunohistochemistry IHNV Infectious Haematopoeitic Necrosis Virus IP intra-peritoneal IPNV infectious pancreatic necrosis virus ISAV Infectious Salmon Anaemia Virus M Molar mg milligram ml millilitre mM millimolar ng nanogram Os Out Skerries IPNV isolate PBS phosphate buffered saline PBSN PBS thiomersol with 1 % sodium azide PBST PBS-Tween20 PCR polymerase chain reaction PCV packed cell volume Pg picogram PIT tags passive integrated transponders PNNV Piscine Nervous Necrosis Virus (Nodavirus) RFLP restriction fragment length polymorphism RNA ribonucleic acid RTG-2 rainbow trout gonad cells RT-PCR reverse transcription PCR SV2 salmon advanced to go to sea within 6 months SAPU Scottish Antibody Production Unit SERAD Scottish Executive Rural Affairs Department SHK Salmon Head Kidney cells SPB sodium phosphate buffer SSN-1 Striped Snakehead fry/peduncle cells ssRNA single stranded RNA TBE tris-borate EDTA buffer TBS tris buffered saline TCID tissue culture infective dose TE tris-EDTA buffer TMB 3,3’, 5,5’, tetramehyl-benzidine dihydrochloride TNE tris-NaCl-EDTA buffer TSA tryptic-soya agar UV ultra-violet VNTR variable number tandem repeats Table of Contents Declaration.............................................................................................. .............................. i Abstract ..................................................................................................................................ii Acknowledgements ........................................................................................................... iv Abbreviations ........................................................................................................................v Table of Contents ............................................................................................................
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