STUDIES ON THE CHARACTERISATION AND DETECTION OF Piscirickettsia salmonis By Sema Yuksel BSc Aquaculture, MSc Living Marine Resources Thesis submitted for the degree of Doctor of philosophy University of Stirling 2003 To Tayfun and my family for their endless love and support DECLARATION I hereby declare that this thesis has been composed entirely by myself and is the result of my own investigations. It has not been previously submitted or accepted for any other degrees. Serna Yuksel I ACKNOWLEDMENTS I would like to express my sincere gratitude to the following people for their contribution in the completion of this thesis. First of all, I would like deeply thank to my supervisors, Dr. Kimberly D. Thompson, Dr. Alexandra Adams and Dr. Anthony Ellis, for their continuing support, encouragement, valuable guidance, endless help and kindness throughout the period of my study. My special gratitude is given everyone in Institute of Aquaculture who provided valuable help and advise through these studies, especially to Miss Fiona Muir, Mr. Linton Brown, Dr. Margaret Caimey, Dr. John Taggart, Prof. Hugh W. Ferguson, Dr. William Starkey, Mr. Richard Collins, Mrs. Gillian Dreczkowski, Dr. Janet A. O’Flynn, Dr. David John Morris, Mr. Iain Elliot, Mrs. Debbie Faichney, Mrs. Maureen Menzies, Mrs. Hilary McEwan, Mrs. Karen Snedden, Mr. Stuart D. Millar, Dr. Marianne D. Pearson, Mr. James Dick, Mr. Alan Porter , Mr. Niale Avchinachie and Dr. Ruth Campell. Also many thanks to everyone at the Animal House Unit. I would like also thank to Dr. Roy Palmer and everyone in Aquatic Veterinary Group, National Diagnostics Centre, University of College Ireland for their valuable time to help me learn the cell culture techniques and IFAT. Many thanks to everyone in FRS Marine Laboratory in Aberdeen for their help in carrying out the experimental challenges, especially to Mr. Benjamin E. Williamson in Behaviour and Aquarium Services. Many special thanks go to all of my friends for being supportive and for making my time in Stirling memorable, especially to Emine Turgut, Nirattisai Petchsupa, David Miles, Ioannis Vatsos, Nelly Isyagi, James Neary, Suppalak Puttinaowarat, Md. Ali Reza Faruk, Manual Fuentes, Nick Taylor and all my housemates in Fairview. My love goes to my husband Tayfun Yuksel. Thanks for his endless love, support and patience. Many special thanks to my family for being supportive and always being there for me. I am truly indebted to Turkish Government and Aquaculture Vaccines Limited (AVL) for providing financial support for my studies. II ABSTRACT Piscirickettsia salmonis, the aetiological agent of piscirickettsiosis, has recently been responsible for significant disease outbreaks in a variety of economically important freshwater and seawater fish species cultured worldwide. The development of effective control strategies for the disease has been limited due to a lack of knowledge about the physiology, intracellular growth, transmission and pathogenesis of the organism. It is also notknown how P. salmonis isolates differ when isolated from different locations and from different fish species. The aim of the present study was to characterise phenotypic and serological differences between various P. salmonis isolates. The growth of these isolates in vitro was also examined together with their virulence and their pathogenesis. The antibody response of fish to live and killed P. salmonis was examined in an attempt to understand the antigenicity of the organism, and various diagnostic techniques were developed as a means of controlling the disease. A simple and effective method for the purification of P. salmonis from fish cells to examine the antigenic properties of P. salmonis was developed. P. salmonis purified using differential pelleting and 30% Percoll (v/v) gradient gave yields with the highest purity and the highest infectivity. Fish cell lines were used to examine growth characteristics of P. salmonis isolates in vitro. It appears that P. salmonis has a 3-5 days (d) lag-phase and an 8 d log-phase of exponential growth in CHSE-214 cells. When the susceptibility of different fish cell lines to different P. salmonis isolates were compared, the highest TCID50 ml-1 was obtained in CHSE- 214, SHK-1 and EPC cells. The antigenic structure of the ten P. salmonis isolates was analysed using SDS-PAGE. P. salmonis isolates shared many bands but did appear to have differences in the low molecular weight regions of their profiles. Silver staining of Proteinase-K digested P. salmonis isolates showed that all isolates contained carbohydrate moieties below 30 kDa. Further characterisation of these was performed using a glycoprotein determination kit and a number of different biotin-labelled lectins. The SDS-PAGE profile of isolate R-29 appeared different when passaged three times through fish cell lines. The bacteria grown in CHSE-214, EPC and SBL cells, which showed a higher susceptibility to the bacterium, had more material in the lower region of the gel than bacteria recovered from less susceptible BF-2 and RTG-2 cells where the bands in this region had disappeared. A nested polymerase chain reaction (N-PCR) was used to differentiate different P. salmonis isolates. Isolate R-29 was the only isolate which resulted in a PCR product different to that of the other isolates examined. Ill A whole cell preparation of purified P. salmonis (type strain LF-89) was used to prepare a rabbit polyclonal antibody and six monoclonal antibodies. These antibody probes were used to compare the antigenicity between P. salmonis isolates. Major antigens of P. salmonis were observed at 95, 72, 60, 36, 32 and 20 kDa in Western blot (WB) analysis. The antibodies were also used to develop a variety of antibody-based tests for the more specific determination of the pathogen. A number of staining methods was also tried in an attempt to establish a rapid diagnostic method for piscirickettsiosis. The virulence of P. salmonis isolates was compared in an experimental infection of Atlantic salmon. It was possible to re-isolate the bacterium from infected kidney tissue during the first 4 weeks post-inoculation (wpi) and to detect by ELISA in the kidney of fish sampled 8 wpi. The histopathology observed was similar to that seen during natural outbreaks of piscirickettsiosis. No obvious difference was seen in the pathology between the different isolates used in the experimental infection. Atlantic salmon were immunised with heat-killed preparation of various P. salmonis isolates. The highest antibody response was obtained in sera raised against P. salmonis isolate R-29. This was also found to be the case in fish infected with live R-29 compared with other P. salmonis isolates. Sera from fish either challenged with live bacteria or immunised with heat-killed bacteria were examined in WB analysis. Proteins between 30-60 kDa appeared to be recognised with sera raised against live P. salmonis, unlike the sera raised against heat-killed P. salmonis, except for a strong reaction with the band seen at 60 kDa with sera from fish immunised with isolate R-29. Other bands at 95,72 and 20 kDa were recognised by the fish sera raised against either heat-killed P. salmonis or the live bacteria. For the first time, phage particles were observed to be associated with rickettsia infecting fish. Attempts were made to identify and characterise the phages in this study with a view to using them in the control of P. salmonis. The significance of the characterisation of P. salmonis isolates and the development of tests to detect and identify the pathogen carried out in this study is discussed within this thesis, together with further research which may lead to the development of successful control strategies for piscirickettsiosis. IV ABBREVIATIONS Mg Microgram /il Microlitre ¡im Micrometre /¿M Micromolar 16S rDNA 16 small subunit ribosomal deoxyribonucleic acid 16S rRNA 16 small subunit ribosomal ribonucleic acid 23 S rDNA 23 large subunit ribosomal deoxyribonucleic acid 23S rRNA 23 large subunit ribosomal ribonucleic acid ABP Avidin-biotin-peroxidase complex ATCC American Tissue Culture Collection bp Base pair BF-2 Bluegill, Lepomus macrochirus Rafinesque fry cell line BSA Bovine serum albumine BS-1 Bandeiraea simplicifolia CHSE-214 Chinook salmon, Oncorhynchus tshawytscha embryo cell line Con A Jack bean (Canavalia ensiformes) CPE Cytopathic effect cpm Count per minute CSS Chromogenic substrate solution CTA Coral tree (Erythrina cristagalli) d Days DBI Dot blot immunobinding assay dpi Days post-inoculation dpif Days post-infection DMDS Diatrizoate meglumine and diatrizoate sodium DMEM Dulbecco’s Modified Eagle’s Medium DMSO Dimethyl sulfoxide DNA Deoxyribonucleic acid EDTA Ethylenediaminetetraacetic acid ELISA Enzym-linked immunosorbent assay EM Electron microscopy EPC Epithelioma papulosum cyprini Cyprinus carpió cell line FAT Fluorescent antibody technique V FBS : Foetal bovine serum FCS : Foetal calf serum FITC : Fluorescein isothiocyanate h : Hours HAT : Hypoxanthine aminopterin thymidine media supplement H&E : Haematoxylin and Eosin HGA : Horse gram (Dolichos biflorus) HRP : Horseradish peroxidase HSWB : High salt wash buffer Hsp : Heat shock protein IFAT : Indirect fluorescent antibody technique ICU : Infected cell counting units IgG : Immunoglobulin G IGS : Immuno-gold staining m e : Immunohistochemistry M : Intramuscular IOA : Institute of Aquaculture, University of Stirling IP : Intraperitoneal US : Internal transcribed spacer IU : International units IV : Intravaneous kbp : Kilo base