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Stress responses of juvenile salmonids to immunological challenge Ackerman, Paige Adrienne 2004

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STRESS RESPONSES OF JUVENILE SALMON IDS TO IMMUNOLOGICAL CHALLENGE by Paige Adrienne Ackerman B.Sc, The University of British Columbia, 1991 M.Sc, The University of British Columbia, 1995 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY In THE FACULTY OF GRADUATE STUDIES (Department of Animal Science) We accept this degree as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA July 2004 © Paige Adrienne Ackerman, 2004 ii Abstract Physiological and cellular stress responses of fish to immunological challenge were examined using an experimental approach involving whole animal and tissue culture. The first study examined effects of sub-lethal ammonia exposure on stress responses and disease susceptibility of salmonids. In seawater at a pH of 7.8, salinity of 30 g/kg, and temperature of 10-12°C, the Environmental Protection Agency (EPA) defines acute ammonia exposure in terms of a Criteria Maximum Concentration (CMC) of 23 mg Total Ammonia Nitrogen (TAN)/L and chronic exposure as a Criteria Continuous Concentration (CCC) of 3.4 mg TAN/L. Juvenile chinook salmon (Oncorhynchus tshawytscha) were exposed to two sub-lethal concentrations (10.0 mg TAN/L and 2.5 mg TAN/L) for a period of 10 d. Samples taken during the exposure indicate that at levels well below EPA guidelines, metabolic changes occurred that were indicative of a physiological stress response. A subsequent disease challenge using the marine pathogen Vibrio anguillarum resulted in a significant increase in mortality of fish exposed to 10 mg TAN/L compared to controls. Mortality of fish exposed to 2.5 mg TAN/L was between that of controls and fish exposed to the higher concentration. The cellular stress response as measured by heat shock protein 70 (hsp70) changed in relation to exposure concentration. Hsp70 increased in liver tissue in fish exposed to 2.5 mg TAN/L but both concentrations resulted in hsp70 decrease in head kidney, although this was only significant in fish exposed to 10.0 mg TAN/L. Results suggest that current environmental standards do not protect fish from an immunological point of view. To further investigate the relationship between hsp70 levels and disease, juvenile rainbow trout (O. mykiss) were experimentally infected with the pathogen V. anguillarum. Changes in blood pathogen levels and corresponding indicators of physiological and cellular stress were examined over a period of 8 d. Chloride ion concentrations significantly decreased by 6 d post-challenge. At this point blood pathogen concentrations had reached peak levels. Haematocrit and haemoglobin levels also changed over time. The decrease in plasma protein concentration at 6 d post challenge, indicating a probable haemodilution, was a causative factor in these changes. Plasma Cortisol and liver and head kidney hsp70 levels increased from day 3, reaching peak values 24 h prior to clinical signs of disease. Hsp70 is involved in the glucocorticoid response and the observed increase of both hsp70 and Cortisol may have functional consequences in the immune response. A simple explanation for responses to live challenge may be that infectious bacteria damage cellular components, thus altering cellular homeostasis and inducing stress proteins. Ill To investigate the role played by actions of a live pathogen versus host responses to an inanimate antigen, subsequent experimental challenges were performed over seven days using the following treatments: 1) a bacterin based on killed V. anguillarum, 2) microbial lipopolysaccharide (LPS) from Escherichia coli, and 3) saline sham injection. Levels of hsp70 differed in the two tissues tested. In head kidney tissue, LPS treatment resulted in an increase in hsp70 3 d post injection while, in liver, both test treatments resulted in a decrease in hsp70 after 2 d. Elevated plasma Cortisol was observed only in fish injected with LPS; this response was transient and returned to control levels by day 3 suggesting that it is endotoxin that is at least partially responsible for physiological responses to infection. To separate effects of Cortisol from hsp70 responses, the antagonistic steroid analogue RU-486 was used to block Cortisol receptors and therefore physiological responses to Cortisol. Fish received one of four treatments: 1) oil carrier + saline; 2) RU-486 in oil carrier + saline; 3) oil carrier + V. anguillarum challenge; or 4) RU-486 in oil carrier + V. anguillarum. Fish receiving the blocker in conjunction with disease challenge had a significant increase in plasma Cortisol at most time points compared with all other groups. These same fish had a significant decrease in liver hsp70 early on during challenge and a trend towards greater susceptibility to disease challenge. Previous studies with fish suggest that RU-486 does not affect circulating levels of Cortisol but the current study found that levels were increased over controls even in the absence of a pathogen so, although physiological effects may have been blocked, accumulation of the hormone was not. To examine this further, a subsequent study investigated effects of an immunological challenge at the cellular level and effects of Cortisol during such challenge. Two cell cultures were utilized to investigate the following treatments, both with and without a physiological concentration (300 ng/mL) of exogenous Cortisol: 1) saline; 2) E.coli LPS; 3) V. anguillarum LPS; 4) killed V. anguillarum; and 5) live V. anguillarum. When exposed to E. coli LPS, an Atlantic salmon [Salmo salar) head kidney (SHK-1) derived cell line had a greater hsp70 level at 4 h than 24 h but this difference was abolished by addition of Cortisol. There was no statistical difference between 4 and 24 h levels in SHK-1 cells exposed to V. anguillarum LPS alone, although the trend was similar to that seen in cells exposed E. coli LPS. When Cortisol was combined with this treatment the difference between the two times was significant. The addition of Cortisol resulted in a similar decrease in hsp70 levels between 4 and 24 h in cells exposed to either killed or live pathogen. This may indicate that an attenuating/dampening effect similar to that which has been previously reported may have occurred. Primary cultures of hepatocytes obtained from rainbow trout (O. mykiss) responded quite differently to iv challenge. Although E. coli LPS exposure still resulted in a significant increase in hsp70, it did so both in the presence and-absence of Cortisol, and responses were greater at 24 h than 4 h. Hepatocytes incubated with LPS from V. anguillarum also had an increase in hsp70 between 4 and 24 h. Addition of Cortisol did not result in an attenuation of the hsp70 response. Rather, the presence of Cortisol resulted in increased hsp70 in all groups from 4 to 24 h. A clear difference between tissues was demonstrated but cells responded in a fashion that was opposite to that which was observed in whole animal studies. My thesis research provides new insights into physiological processes that underlie an immunological response. Many studies have observed effects of stress on disease resistance and immunological function, but few have examined physiological responses to immunological challenge. That a host of physiological stress responses to immune challenge were observed and that these responses vary so clearly between whole animal and isolated cell cultures underscores the need for further studies to better understand relationships between physiological stress responses and the immune system. Table of Contents Abstract ii Table of Contents v List of Tables vii List of Figures ix Preface xii Acknowledgements xiii General Introduction and Thesis Overview '. 1 The Physiological Stress Response 1 The Cellular Stress Response 3 The Fish Immune System 4 Relationships Between Stress and Immune Function 5 Thesis Objectives and Overview 9 References 11 Section I. Stress responses of juvenile salmonids to external and internal challenge 15 CHAPTER 1 Effects of sub-lethal environmental ammonia exposure on recently saltwater acclimated juvenile Chinook salmon (Oncorhynchus tshawytscha) 16 Introduction 16 Materials and Methods 17 Results ; 21 Discussion 37 References 42 CHAPTER 2 Physiological and cellular stress responses of juvenile rainbow trout (Oncorhynchus mykiss) to vibriosis 46 Introduction 46 Materials and Methods 47 Results and Discussion 49 References 61 Section II. The effect of non-lethal immune challenge on stress responses 64 CHAPTER 3 Physiological and cellular stress responses of juvenile rainbow trout (Oncorhynchus mykiss) to LPS and a Vibrio anguillarum bacterin 65 Introduction 65 Methods and Materials 66 Results 68 Discussion 81 References 85 vi Section III. The effect of Cortisol increase during infection 88 CHAPTER 4 Use of the Cortisol blocker RU-486 in fish health studies 89 Introduction 89 Methods and Materials 90 Results 92 Discussion 107 References 110 Section IV. Cell culture studies on the cellular stress response to immunological challenge during exposure to physiological levels of Cortisol 112 CHAPTER 5 The cellular stress responses of primary cultured rainbow trout hepatocytes and a macrophage-like Atlantic salmon cell line (SHK-1) to experimental immunological challenge 113 Introduction 113 Methods and Materials 115 Results 118 Discussion 127 References 131 GENERAL DISCUSSION 134 Comments on Future Research 137 References 139 VII List of Tables Table 1-1. Mean (± SE) plasma Cortisol, plasma protein, haematocrit, erythrocytes and nitroblue tetrazolium (NBT) positive cells of rainbow trout experimentally exposed to three concentrations of environmental ammonia as total ammonia nitrogen (TAN). Superscript letters indicate significant differences between groups at each sample time. Numbers denote differences within groups across sample times (P < 0.05). Superscript letters within the treatment column indicate a significant difference between dose effects 33 Table 1-2. Mean (± SE) plasma glucose, plasma lysozyme activity, haemoglobin, liver and head kidney hsp70, and leucocyte numbers of rainbow trout experimentally exposed to three concentrations of environmental ammonia as total nitrogen (TAN). Superscript letters indicate significant differences between groups at each sample time, and numbers denote differences within groups across sample times (P < 0.05). Superscript letters within the treatment column indicate a significant difference between dose effects 34 Table 1-3. Mean (± SE) lymphocyte and neutrophil numbers of rainbow trout experimentally exposed to three concentrations of environmental ammonia as total nitrogen. Superscripts indicate significant differences between groups at each sample time (P < 0.05). Superscript letters within the treatment column indicate a significant difference between dose effects 35 Table 1-4. Mean (± SE) thrombocyte and monocyte numbers of rainbow trout experimentally exposed to three concentrations of environmental ammonia as total nitrogen (TAN). Superscripts indicate significant differences between groups at each sample time (P < 0.05). Superscript letters within the treatment column indicate a significant difference between dose effects 36 Table 2-1. Mean (± SE) haemoglobin and haematocrit in rainbow trout experimentally infected with V. anguillarum. Asterisks indicate significant differences between the control and infected groups (haemoglobin P = 0.002 ; haematocrit P = 0.001) (n = 6) at each sample time. C = Controls ; E = Experimentally infected 58 Table 2-2. Mean (± SE) plasma variables of rainbow trout experimentally infected with V. anguillarum. Asterisks indicate significant differences between the control and infected groups at each sample time (P < 0.001) (n = 6). C = Controls ; E = Experimentally infected ; 59 Table 2-3. Mean (± SE) plasma ion concentrations of rainbow trout experimentally infected with V. anguillarum. Asterisks indicate significant differences between the control and infected groups at each sample time (P < 0.001) (n = 6). C = Controls ; E = Experimentally infected. 60 Table 3-1. Mean (± SE) plasma concentrations of protein, glucose, lysozyme and Cortisol from juvenile rainbow trout challenged with saline, 35 mg/kg E. coli LPS, or 10 6 cells/fish formalin killed V. anguillarum. Superscript letters denote differences between treatments within sampling time periods (P<0.05, n=10) 77 Table 3-2. Mean (± SE) liver and head kidney tissue levels of hsp70 from juvenile rainbow trout challenged with saline, 35 mg/kg E. coli LPS, or 10 6 cells/fish formalin killed V. anguillarum. Superscript letters denote differences between treatments within sampling time periods (P<0.05, n=10) 78 VIII Table 3-3. Mean (± SE) haematology of juvenile rainbow trout challenged with saline, 35 mg/kg E. coli LPS, or 10 6 cells/fish formalin killed V. anguillarum. Superscript letters denote differences between treatments within sampling time periods, superscript numbers indicate differences within treatments between sampling times (P<0.05, n=10) 79 Table 3-4. Mean (± SE) haematology of juvenile rainbow trout challenged with saline, 35 mg/kg E. coli LPS, or 10 6 cells/fish formalin killed V. anguillarum. Superscript letters denote differences between treatments within sampling time periods, superscript numbers indicate differences within treatments between sampling times (P<0.05, n=10) 80 Table 4-1 . Plasma glucose, Cortisol and protein concentrations and lysozyme activity in juvenile rainbow trout sham challenged or disease challenged (V. anguillarum) following implantation with oil or RU-486 in an oil carrier. Superscript letters denote differences +/- 1 SE between treatments within sampling time periods, numbers indicate differences ± SE within treatment between sampling times (P<0.05, n=10) 103 Table 4-2. Haematology (haematocrit, haemoglobin and erythrocyte numbers) of juvenile rainbow trout sham challenged or disease challenged (V. anguillarum ) following implantation with oil or RU-486 in an oil carrier. Superscript letters denote differences +/- 1 SE between treatments within sampling time periods, numbers indicate differences ± SE within treatment between sampling times (P<0.05, n=10) 104 Table 4-3. Blood cell concentrations in juvenile rainbow trout sham challenged or disease challenged (V. anguillarum ) following implantation with oil or RU-486 in an oil carrier. Superscript letters denote differences ± SE between treatments within sampling time periods, numbers indicate differences +/- 1 SE within treatment between sampling times (P<0.05, n=10) 105 Table 4-4. Tissue hsp70 levels in juvenile rainbow trout sham challenged or disease challenged (V. anguillarum ) following implantation with oil or RU-486 in an oil carrier. Superscript letters denote differences ± SE between treatments within sampling time periods, numbers indicate differences +/- 1 SE within treatment between sampling times (P<0.05, n=10) 106 Table 5-1. LDH Activity in SHK-1 and hepatocyte cultures following experimental exposure to immunological challenge in the absence and presence of exogenous Cortisol (300 ng/mL). Superscript letters denote differences (± SE) between treatments within sampling time periods (4 or 24 h), numbers indicate differences (± SE) within treatment between 4 and 24 h sampling times (P<0.05, n=10) 126 ix List of Figures Figure 1-1. Plasma ammonia concentration in juvenile Chinook smolts exposed to three levels of environmental ammonia as total ammonia nitrogen (TAN). Means are presented +/- SE. Superscripts denote statistical difference by two way ANOVA (P < 0.05) within each sampling period only 25 Figure 1-2. Plasma Cortisol concentrations of juvenile chinook smolts exposed to three levels of environmental ammonia as total ammonia nitrogen (TAN). Means are presented ± SE. Superscripts denote statistical difference by two way ANOVA (P < 0.05) within each sampling period only 26 Figure 1-3. Plasma glucose concentrations of juvenile chinook smolts exposed to three levels of environmental ammonia as total ammonia nitrogen (TAN). Means are presented ± SE. Superscripts denote statistical difference by two way ANOVA (P < 0.05) within each sampling period only 27 Figure 1-4. Plasma lysozyme activity in juvenile chinook smolts exposed to three levels of environmental ammonia as total ammonia nitrogen (TAN). Means are presented ± SE. Superscripts denote statistical difference by two way ANOVA (P < 0.05) within each sampling period only 28 Figure 1-5. Liver hsp70 concentrations in juvenile chinook smolts exposed to three levels of environmental ammonia as total ammonia nitrogen (TAN). Means are presented ± SE. Asterisks denote statistical differences by two way ANOVA (P < 0.05) within each sampling period only 29 Figure 1-6. Head kidney hsp70 concentrations in juvenile chinook smolts exposed to three levels of environmental ammonia as total ammonia nitrogen (TAN). Means are presented ± SE. Asterisks denote statistical differences by two way ANOVA (P < 0.05) within each sampling period only 30 Figure 1-7. Concentration of nitro blue tetrazolium (NBT) positive cells at 6 and 244 h during exposure to three levels of ammonia as total ammonia nitrogen (TAN). Means are presented ± SE. Asterisk denotes statistical difference within each sampling period only (P < 0.05) 31 Figure 1-8. Cumulative mortality in three groups of juvenile chinook smolts challenged with105 cfu/fish L. anguillarum following a 244 h exposure to three levels of environmental ammonia as total ammonia nitrogen (TAN) 32 Figure 2-1. Mean (+ SE) viable L. anguillarum in colony forming units per mL of blood and cumulative percent mortality for juvenile rainbow trout challenged with L. anguillarum by intraperitoneal injection (9.196 x 103 cells/fish). Significant differences in blood pathogen counts between days are noted by different letters (P < 0.001) 54 Figure 2-2. Viable L. anguillarum in blood and plasma Cortisol concentration following injection challenge into juvenile rainbow trout. Means are presented ± SE. Significant differences in blood pathogen counts between days are indicated by different letters (P < 0.001). Significant plasma Cortisol level differences between control and infected fish are noted with asterisks (P < 0.001) 55 X Figure 2-3. Hsp70 levels in liver of juvenile rainbow trout experimentally infected with L. anguillarum compared with sham injected controls. Means are presented ± SE. Significant differences between control and infected measures are denoted by asterisks (P < 0.0001). ..56 Figure 2-4. Mean (+ SE) hsp70 levels in liver of juvenile rainbow trout experimentally infected with L. anguillarum compared with sham-injected controls. Significant differences between control and infected measures are denoted by asterisks (n = 6, P < 0.0001) 57 Figure 3-1. Mean (± SE) plasma protein concentration over 7 days following injections with a formalin killed bacterin, 35 mg/kg LPS, or saline. Statistical differences between groups within time periods are denoted by superscripts (P < 0.05) 70 Figure 3-2. Mean (± SE) plasma lysozyme activity (B) over 7 days following injections with a formalin killed bacterin, 35 mg/kg LPS, or saline. Statistical differences between groups within time periods are denoted by superscripts (P < 0.05) 71 Figure 3-3. Mean (± SE) plasma Cortisol concentration over 7 days following injections with a formalin killed bacterin, 35 mg/kg LPS, or saline. Statistical differences between groups within time periods are denoted by superscripts (P < 0.05) 72 Figure 3-4. Mean (± SE) plasma glucose concentration over 7 days following injections with a formalin killed bacterin, 35 mg/kg LPS, or saline. Statistical differences between groups within time periods are denoted by superscripts (P < 0.05) 73 Figure 3-5. Mean (± SE) erythrocyte numbers over 7 days following injections with a formalin killed bacterin, 35 mg/kg LPS, or saline. Statistical differences between groups within time periods are denoted by superscripts (P < 0.05) 74 Figure 3-6. Mean (± SE) hsp70 levels in head kidney tissue over 7 days following injections with a formalin killed bacterin, 35 mg/kg LPS, or saline. Statistical differences between groups within time periods are denoted by superscripts (P < 0.05) 75 Figure 3-7. Mean (± SE) hsp70 levels in liver tissue over 7 days following injections with a formalin killed bacterin, 35 mg/kg LPS, or saline. Statistical differences between groups within time periods are denoted by superscripts (P < 0.05) 76 Figure 4-1 . Percent cumulative mortality in juvenile rainbow trout challenged with104 cfu/fish L. anguillarum in the presence and absence of the Cortisol blocker RU-486 95 Figure 4-2. Plasma Cortisol concentration in fish challenged with and without RU-486 and L. anguillarum. Means are presented ± SE. Letters denote statistical differences within each sampling period (P < 0.05) 96 Figure 4-3. Plasma protein concentration in fish challenged with and without RU-486 and L. anguillarum. Means are presented ± SE. Letters denote statistical differences within each sampling period (P < 0.05) 97 Figure 4-4. Haemoglobin concentrations in fish challenged with and without RU-486 and L. anguillarum. Means are presented ± SE. Letters denote statistical differences within each sampling period (P < 0.05) 98 xi Figure 4-5. Haematocrit concentrations in fish challenged with and without RU-486 and L. anguillarum. Means are presented ± SE. Letters denote statistical differences within each sampling period (P < 0.05) 99 Figure 4-6. Erythrocyte concentrations in fish challenged with and without RU-486 and L. anguillarum. Means are presented ± SE. Letters denote statistical differences within each sampling period (P < 0.05) 100 Figure 4-7. Liver hsp70 tissue levels in fish challenged with and without RU-486 and L. anguillarum. Means are presented ± SE. Letters denote statistical differences within each sampling period (P < 0.05) 101 Figure 4-8. Head kidney hsp70 tissue levels in fish challenged with and without RU-486 and L. anguillarum. Means are presented ± SE. Letters denote statistical differences within each sampling period (P < 0.05) 102 Figure 5-1. Lysozyme activity in SHK-1 cells at 4 and 24 h following challenge in the presence or absence of Cortisol (300 ng/mL). Means are presented ± SE. Asterisks denote statistical differences between sampling periods within treatment between 4 and 24 h only (P < 0.05).120 Figure 5-2. Lysozyme activity in SHK-1 cells at 4 and 24 h following challenge in the presence or absence of Cortisol (300 ng/mL). Means are presented ± SE. Letters denote statistical differences between treatments within each sampling time period (4 or 24 h) only. (P < 0.05). 121 Figure 5-3. Hsp70 in SHK-1 cell culture at 4 and 24 h following challenge in the presence and absence of Cortisol (300 ng/mL). Means are presented ± SE. Asterisks denote statistical differences between sampling periods within treatment between 4 and 24 h only (P < 0.05).122 Figure 5-4. Hsp70 in SHK-1 cell culture at 4 and 24 h following challenge in the presence and absence of Cortisol (300 ng/mL). Means are presented ± SE. Letters denote statistical differences between treatments within each sampling time period (4 or 24 h) only (P < 0.05). 123 Figure 5-5. Hsp70 in hepatocyte primary cell culture at 4 and 24 h following challenge in the presence and absence of Cortisol (300 ng/mL). Means are presented ± SE. Asterisks denote statistical differences between sampling periods within treatment between 4 and 24 h only (P < 0.05) 124 Figure 5-6. Hsp70 in hepatocyte primary cell culture at 4 and 24 h following challenge in the presence and absence of Cortisol (300 ng/mL). Means are presented ± SE. Letters denote statistical differences between treatments within each sampling period (4 or 24 h) only (P < 0.05) 125 XII Preface This thesis has been written in a manuscript based format according to the University of British Columbia guidelines. Chapter Two has been published; Ackerman, P.A., and G.K. Iwama. 2001. Physiological and Cellular Stress Responses of Juvenile Rainbow Trout to Vibriosis. Journal of Aquatic Animal Health. 13:173-180. All of the work was performed by myself. Chapter One represents a portion of a collaborative effort with Dr. B.J. Wicks (Department of Zoology, University of British Columbia) and Dr. D.J. Randall (Department of Biology and Chemistry, City University of Hong Kong). Dr. Wicks performed the ammonia exposure and ammonia measurements. All data analysis other than water and plasma ammonia concentration within this section was performed by me. Culture of SHK-1 cells outlined in Chapter Five was performed by Ms. Sandra Sperker (Institute of Marine Biosciences, National Research Council of Canada) and isolation of hepatocytes also described in this Chapter was performed by Ms. Anne Todgham (Faculty of Agricultural Sciences, University of British Columbia). All other cell manipulation and subsequent data analysis was carried out by me. There has been some debate recently about the classification of the pathogen used throughout this thesis. When I commenced this research the bacteria was classified as Vibrio anguillarum, it was subsequently reclassified as Listonella anguillarum but there has been some resistance to this in many quarters, so within the context of this thesis I have chosen to revert to use of the classification Vibrio anguillarum. XIII Acknowledgements How does one say goodbye to something that has been such a huge part of their life. I truly don't know how. It was never my personal mission to achieve the rank of "longest resident of the Iwama Lab", yet 13 years after walking through the door I find myself in that not so enviable position. Not many students can claim that they outlasted their supervisor. I wish to thank George Iwama for opening the door so many years ago and giving me the opportunity and support to explore my interests through the completion of my earlier MSc thesis as well as this PhD. I am grateful for the many opportunities given to me during my residence in the lab. Thank you also for the gift of your friendship. I would like to also thank Dave Randall, Alec Maule, Chris Bayne, Martin Adamson and Kim Cheng for providing guidance on the preparation the research proposal that has guided me through the past years and for providing valuable comments during the preparation of this thesis. Financial support for this research was provided by NSERC and AquaNet grants to Dr. Iwama, and an NSERC postgraduate scholarship, a Science Council of BC GREAT scholarship, a University of BC graduate fellowship, and a Faculty of Agricultural Sciences Howland fellowship to myself. I am grateful to have made so many lasting friendships through my years in the Iwama Lab. That web is wide and I consider myself blessed to have been a part of it. Many of the Iwama'ites who went before me helped me achieve my goals and continue on when I no longer thought I could. Thank you to Shannon Balfry who was always there to prop me up when I needed it. To John Morgan, thanx for all those "cyber-chats" that helped me through the difficult times; you never lost faith in me even when I'd lost it in myself. There are many others who helped me in so many ways during this thesis and my time in the lab. In particular I would like to thank Ellen Teng (it was never the same without you here), Rob Forsyth (my ELISA quality control resource), Grace Cho (more moral support) Kazumi Nakano, Alberto Schlicht, Rosalind Leggatt, Anne Todgham, Bev Wicks (we did sort of adopt you) and Dominic Toa. I thank you all. There are many others outside of the lab who I would like to thank as well. Glenn and Kellyanne: Koerners was always so much more than just a beer or five on Fridays; thank you both for providing sanity at the end of every week. Lorraine and Derek, you thought I was insane when I left to go back to school, but the experiences I gained through you gave me the ability to stay grounded and deal with situations no student needs to contend with. And of course I must recognize all the fish whose lives were sacrificed in the studies in the hopes that someday the research I have carried out may be of some benefit. Finally, I would like to acknowledge the support of my family. To my parents, Wayne and Margaret Ackerman, thank you for never holding me back. Although you never really understood, you knew enough to let me go. I could not have completed this thesis without moral support of Kirk Kohn. You are the love of my life and the best friend I have ever had. You provided me with unending love and patience as I struggled down this long and winding road, and a struggle it has been. Without you I never would have survived. I'm not quite certain how you managed to survive me. "This vain presumption of understanding everything can have no other basis than never understanding anything. For anyone who had experienced just once the perfect understanding of one single thing, and had truly tasted how knowledge is accomplished, would recognize that infinity of other truths of which he understands nothing". - Galileo Galilei (1564-1642) To the end of an era (1987 - 2002) - Viva Ichthyo Voce 1 GENERAL INTRODUCTION AND THESIS OVERVIEW Each day we are on a battlefield. Our bodies encounter a multitude of potential invaders in the form of microorganisms and the presence of a functional immune system is the foundation for our protection against them. In order to survive, we not only require appropriate weapons, but we must be able to produce them rapidly enough and in a great enough quantity to protect us before the system is overwhelmed. Ideas abound regarding how to fix health, but the fact remains that the immune system is the basis for such repair and a healthy physiology is necessary for its effective functioning. If we could truly understand the systems, we could make them work when and how we preferred. One of the most efficient methods to achieve this is vaccination, but in order to formulate an effective vaccine, we need to understand relationships between host and pathogen. Simply assuming that a vaccine is suitable because it reduces disease incidence is inappropriate because such assumptions do not address underlying more subtle responses. All the body's systems are intertwined, yet the majority of studies do not examine physiology underlying immune responses, lethal or otherwise. Pathophysiological changes of many mammalian diseases have been described, yet the processes in fish are less well studied. With a worldwide increase in aquaculture, fish health is daily becoming a more important issue and the means to maintain healthy populations, both cultured and wild, is a high priority. Gaining a clearer understanding of the progression of responses that occur within a host during an infection will ultimately lead to more efficient and efficacious preventative measures and treatments. The Physiological Stress Response Stress is a condition experienced by all organisms on a regular basis and is often inappropriately considered to be maladaptive. Physiological variables are a natural part of any organisms' existence; however the degree of stress that an animal endures during daily pressures dictates the severity of responses and whether or not resulting changes will be detrimental to health. Nearly 60 years ago, the terms stress and stress syndrome were introduced following a series of experiments identifying that deleterious stimuli were capable of resulting in pathophysiological changes in rats (Selye 1936). Since then, it has been studied extensively and defined in many ways. Hans Selye (1950) provided a number of these with perhaps the simplest being "the non-specific response of the body to any demand put on it". Selye identified that all activity involves stress and that the stress responses are essential to life. If there are no demands on the body, the body is without life. Selye also indicated that 2 individual stressors are not necessarily harmful; instead it is the build up that occurs when an organism cannot escape the stressor that leads to distress. It is important to note that there are differences within populations or even individuals of a population with respect to stress tolerance (see Iwama ef al. 1999). Further to this, there may be a cross tolerance development between different stress stimuli (Selye 1946) indicating that resistance to stress may be non-specific in many cases. With the interest in stress responses in the following decades, Selye (1950) proposed a general definition that has been widely used in human medicine; "the sum of all the physiological responses by which an animal tries to maintain or reestablish a normal metabolism in the face of a physical or chemical force." This has since been widely adapted to different fields and physiologists have established slightly different definitions to fit individual purposes: "A state produced by environmental or other factors which extends the adaptive response beyond the normal range or disturbs normal functioning so the chances of survival are reduced" (Brett 1958), "The sum of all the physiological responses by which an animal tries to maintain or reestablish normal metabolism in the face of physical or chemical change" (Wedemeyer et al. 1981), "The sum of all physiological responses that occur when animals attempt to reestablish homeostasis, the stressor being an environmental alteration and stress the organism's response" (Wedemeyer et al. 1981), "Alteration of one or more physiological variables to the point that long term survival may be impaired" (Bayne and Zelikoff, 1995). Regardless of the description that one chooses, what stands out clearly from these and other definitions of stress is that it is consistently defined in terms of physiological adaptations to environmental perturbations. It is a state of decreased health induced by any factor that challenges the homeostatic nature of an organism thereby threatening survival by extending normal processes beyond a natural range. Perhaps Selye's simplest definition is the most accurate "the non-specific response of the body to any demand put on it". Three things then become the focus (i) the demands that are placed on the body, (ii) the nature and degree of stress brought about by each or their combination, and (iii) the biological significance of those responses. Selye (1950) also proposed a three stage response to stress referred to as the general adaptation syndrome (GAS). An alarm reaction during which the stress hormones are released characterizes the first stage. The second phase of the response involves resistance to the stressor; the animal attempts to adapt to the state or situation with which it is confronted. Should the stressor prove to be too severe or too long lasting, the third and final stage of the syndrome is exhaustion. When a fish is exposed to a stressor, it will respond with a series of 3 biochemical and physiological changes as it attempts to compensate (reviewed by Wendelaar Bonga 1997). Effects of these compensatory changes have been extensively explored and widely used definitions and descriptions of primary, secondary and tertiary effects of stress in fish exist (Mazeaud et al. 1977; Wedemeyer et al. 1981; Barton 2002). Briefly, during the 'primary response', a stressful event is recognized physiologically and a neuroendocrine/endocrine response is mounted (Gamperl et al. 1994). Cortisol is the hormone most often measured in fish as an indicator of a physiological stress response. The 'secondary response' is characterized by effect of these "stress hormones" on biochemical and physiological systems as the animal attempts to mobilize energy resources to cope with the stressor on a metabolic level. Glucose and glycogen levels are typically measured indicators of such stress-induced metabolic change. Finally, an animal unable to adapt or acclimate to a stressor with which it is confronted may undergo changes on a more visible scale. Decreases in growth, disease resistance, reproductive success, and swimming performance, are classified as 'tertiary responses' and may have influences at a population level, resulting in reduced recruitment and productivity leading to altered species abundance and diversity. The Cellular Stress Response While studying the fruit fly Drosophila busckii, Ritossa (1962) noted a salivary gland chromosomal puffing following a thermal stress. Heat stress induced production of a set of proteins that became universally known as heat shock proteins (hsps) and these have since been shown to be both highly conserved and inducible (Lindquist 1988). While initial studies focused on heat as a stressor, hsps are induced in response to a variety of stressors and environmental contaminants other than thermal shock (Sanders 1993), leading to the use of a perhaps more appropriate term, stress proteins. Hsps are present under normal circumstances in all cells, in all forms of life, at all stages of development. They play important roles in maintenance of homeostasis through protein transport, folding, and degradation (Morimoto et al. 1990), but many other functions are still being elucidated. They act as "chaperones", ensuring that proteins are folded correctly and transported to appropriate cellular compartments for use or destruction. It has been shown repeatedly that a non-lethal pre-treatment induces increased synthesis and this has been correlated with cellular protection against subsequent shock that may have otherwise been lethal (Ciavarra et al. 1990). In addition, protection afforded by one stress may often protect against a different form of stress, providing organisms with what is termed 'cross-protection' 4 (see Lindquist 1988). For example, DuBeau et al. (1998) found that non-lethal heat shock provided juvenile salmon with cross-protection against subsequent severe osmotic challenge. Stress proteins have been closely examined for potential use as biomarkers for environmental monitoring (Sanders 1990; Sanders 1993), although as increasingly more events are demonstrated to induce the response their suitability for identifying specific environmental stressors is questionable (Iwama et al. 2004). A number of hsp families have been identified and they are named, and generally classified, according to their molecular mass (kilodaltons, kDa). These are hsp100, hsp90, hsp70, hsp60 and the low molecular weight hsps (16-30 kDa) (Morimoto et al. 1990). Of these, the most widely studied stress protein is hsp70. The Fish Immune System Fish have defences against pathogenic invasion much like those found in higher vertebrates (Dalmo et al. 1997) and their immune system is similarly divided into two branches. As in higher vertebrates, the immune system recognizes self from non-self and both innate and adaptive immune systems are present. The innate immune system represents the first and second lines of defence against infection. The first line of defence is made up of physical (e.g. epithelial surfaces of the skin, gills and gut), mechanical (e.g. mucus), and chemical (e.g. low pH, digestive enzymes, and mucosal enzymes such as lysozyme) barriers that function to prevent entry of micro-organisms into tissues. If bacteria successfully gain entry, the second line of defence comes into play in an attempt to eliminate pathogens prior to development of disease. Internal innate defences include such biologically active molecules as lysozyme, C-reactive protein and the alternative complement system. These function to destroy invading pathogens directly or opsonize them and enhance cell-mediated defences such as phagocytosis. The inflammatory response is a complex process that is an important component of the second line of defence. It brings together plasma (containing complement proteins, antibodies, etc.) and immunological cells at sites of damage to localize, dilute, and neutralize or destroy an infectious agent. The innate immune system is active from birth/hatch, acts on many organisms without specificity, and does not become more efficient upon subsequent exposure to the same organism. Fish also have a specific (or adaptive) immune system that is characterized by acquired responses against specific foreign antigens. Acquisition of memory is a key feature of this system, allowing it to react more efficiently and rapidly when an agent is encountered a second time. Key cells and molecules of the specific immune system are lymphocytes and 5 antibodies. Antibodies recognize structures (epitopes) on the surfaces of infectious agents and bind them, in turn activating effector mechanisms to eliminate the pathogen from the host. An example of such an effector mechanism is phagocytosis and intracellular killing by macrophages and neutrophils. The classical complement cascade is another example of an effector mechanism brought about by antibody binding. This cascade is responsible for bacterial clearance either through opsonization or through the formation of a membrane attack complex resulting in target cell lysis. Memory gained through survival following an encounter with an infectious agent forms the basis for vaccine use in aquaculture. However, the specific immune response may take days or weeks to mobilize in an ectothermic organism such as a fish and without the protection afforded by constitutive defences an animal would stand little chance against pathogens. For this reason, the innate immune system is generally considered to be the more important system from a natural biological standpoint because it plays such a vital role in controlling an immunological threat until other mechanisms are able to respond. Relationships Between Stress and Immune Function Stress and immune function in fish are related (Maule et al. 1989; Mock et al. 1990; Fevolden et al. 1992; Fevolden et al. 1993; Fevolden et al. 1994), but mechanisms behind the relationship are poorly understood. Cultured salmonids are often exposed to stressors, such as rapid water temperature changes, handling, transportation, confinement, high densities, drugs, environmental toxicants and deterioration of water quality. Wild fish may be subject to such things as industrial effluent and thermal pollution, agricultural runoff or changes in oxygen levels due to algal blooms. All have potential to affect physiological systems of fish in a detrimental manner. Such stressors may raise levels of circulating stress hormones which can affect the immune system and therefore, the ability to resist disease (Maule et al. 1989). Pathogens are ubiquitous; they are in air, in water, in soil and in food and represent one of the most significant naturally occurring biological challenges an organism may encounter. Viruses and bacteria, depending on the region sampled, have been recorded at up to 109/ml_ of seawater (Hennes ef al. 1995). While many studies have examined effects of physiological stress on disease resistance, few have investigated progression of fish diseases from a physiological perspective. Although disease is generally the exception, health remains a major issue in both wild fishery and aquacultural settings where losses due to infection can be substantial and costly. Disease incidences in aquaculture have been reduced by good 6 husbandry practices and vaccines, but there are still pathogens that result in large scale mortalities and for which no cure or viable vaccine exists. It was relatively recent that a natural marriage of the fields of immunology and physiology occurred and the field of immunophysiology emerged. However, the emphasis was still very much on modulatory effects of physiological systems on immune function. More recently, the reverse aspect of the field was emphasized, namely, modulatory effects of the immune system on physiological processes (Plytycz et al. 1995). For example, infection and responses of an animal to that infection can result in a host of changes associated with stress responses that can in turn modify responses to environmental factors. Although we do not generally tend to think of it as such, the immune system can be considered a sensory system that is capable of reacting to stressors in many of the same ways that the physiological systems do (Besedovsky etal. 1975). Plasma Cortisol and glucose levels are widely used indicators of stress in fish (Wedemeyer et al. 1990) and blood levels of both substances generally increase in response to stress (Mazeaud et al. 1977). Elevated blood glucose levels are initiated by the action of adrenaline and sustained through the action of Cortisol presumably to provide an animal with energy to cope with a stressful situation (reviewed by Love 1980). Effects of Cortisol increase on disease resistance have been extensively studied (Robertson et al. 1987; Laidley et al. 1988; Rand et al. 1990; Mesa et al. 1998) but effects of disease on Cortisol production have not been so well characterized and results are sometimes conflicting. Plasma Cortisol is elevated during the latter stages of bacterial kidney disease in juvenile chinook salmon (O. tshawytscha) (Mesa et al. 1998). Robertson et al. (1987) reported a similar increase in Cortisol levels during an unidentified bacterial infection in red drum (Sciaenops ocellatus). The blood haemoflaggelate Cryptobia does not result in a Cortisol increase in rainbow trout (Laidley et al. 1988) nor does a change in plasma Cortisol occur in response to Ichthyoptherius infection (Rand et al. 1990). It is difficult to reconcile these differences without accompanying physiological data to better understand the state of the fish, but conflicting data on Cortisol and disease in fish does underscore the need for more comprehensive descriptions of pathophysiology of fish diseases. Early studies on the hsp response centered on their increase following heat and chemical stressors. There is currently much interest in the roles that hsps may have during an immune response. At the mammalian level, research demonstrated an important role for hsp in the pathology of some inflammatory diseases (Schett et al. 1998) and strong hsp induction elicited by an inflammatory process may protect against auto-oxidative stress brought about by 7 activated immunocytes (reviewed by Jaquier-Sarlin et al. 1994). In mammals, hsp70 is found at higher levels in the phagocytic monocyte/macrophage line of cells than in any other cell type tested, and functions ranging from self/non-self recognition to antigen presentation have been attributed to this family of proteins (reviewed by Bachelet et al. 1998). Bacterial infection resulting in activation of phagocytes and granulocytes results in an increase in bacterial killing activity through mechanisms such as the production of lysozyme, cationic proteins, and reactive oxygen species brought about by respiratory burst activity (i.e. hydrogen peroxide, hydroxyl radical, etc.). These same molecules are also damaging to components of host cells and it has been postulated that one function of hsp production in these cells is to protect against apoptosis arising from auto-oxidation (Bachelet ef al. 1998). Evidence has been found demonstrating that an increase in hsps can lead to an enhancement of immune responses (Mestril ef al. 1994; Rokutan ef al. 1998). While long considered an intracellular protein, hsps also have extracellular functions that are becoming increasingly apparent. Natural destruction of a cell through apoptosis, programmed cell death, does not release "self hsps into circulation, but necrotic cell death does (Basu ef al. 2000). Because these proteins are not normally found outside of the cell, their extracellular presence acts as a proverbial "red flag" indicating that there is a pathophysiological condition somewhere in the body. In mammals such a release of hsps from a necrotic cell leads to nitric oxide production from antigen presenting cells (Panjwani ef al. 2002), cytokine secretion from macrophages and upregulation of several proteins crucial for T-cell activation (Asea et al. 2000; Basu ef al. 2000). The ability of hsps to stimulate T cells and macrophages suggests that they could play a major role in protective immunity (Young 1990). They are often referred to as being "sticky" proteins, as they easily complex to other proteins, both self and non-self. DeNagel et al. (1993) suggest that hsp may play a role in assembly of MHC-class II peptide complexes and subsequent antigen presentation, and members of the hsp70 family may optimize antigen presentation contributing to an efficacious immune response (Mariethoz ef al. 1994). Cell surface receptors for hsps have been identified and are capable of assisting cellular uptake of hsp70-peptide complexes (Castellino ef al. 2000) lending substance to the theory that theyplay roles in the immune system. Hsps themselves are immunodominant antigens produced in large quantities by microbes when they enter the host environment. Immune recognition of pathogen and tumour related hsps has been implicated in serving as an early line of defence for the host organism. Because of their immunogenicity, hsps may have therapeutic application (Edgington 1995) and are being investigated for use against cancerous tumours owing to their apparent functional 8 role in antigen presentation (Blachere ef al. 1993; Harada ef al. 1998). More recent studies have further added to the argument that there is a role of hsps in evolution of the vertebrate immune system (Robert ef al. 2001). These authors show a strong phylogenetic conservation in hsp-antigen presenting cell interactions. Stress proteins could prove to play a very important role in resistance or response to pathogenic infection and this knowledge may prove significant to the aquaculture industry such that vaccines or feeds incorporating hsps as immunomodulators or chemicals that induce their expression could be developed to help reduce disease incidences. Ellis (1981) wrote that "In order to understand and explain the mechanisms and physiological significance of the stress-stressor response phenomenon, biologists need to adopt a multidisciplinary synthetic approach rather than the reductionist one which has proved so successful for understanding mechanistic aspects of biology but is inadequate for explaining the significance of physiological events in functional terms, which is surely, the crux of biology." It seems intuitive that disease should be stressful, yet we have very little information available to us on what is occurring at a physiological level during an immune response and it is worth reiterating that perhaps the ultimate stressor any organism faces is disease. In fish, bacterial kidney disease (BKD) caused by the pathogen Renibacterium salmoninarum resulted in elevated hsp70 levels in clinically infected fish (Forsyth ef al. 1997). BKD is a chronic disease and R. salmoninarum is an ubiquitous organism that has evolved to exist within immunologically active cells. This suggests that the system may not react to the pathogen in an ordinary manner as it does not "see" the threat posed by the pathogen. Hsp70 levels became elevated only after the fish had entered a state of inflammatory response likely due to tissue damage (Forsyth ef al. 1997). Gram-negative pathogens such as Vibrio anguillarum do generate strong and rapid immunological responses in fish and therefore are more likely to result in strong physiological responses. Vaccination against bacterial pathogens may also be physiologically perceived as stressful (Lillehaug ef al. 1992; Ackerman 1995), yet little work has been undertaken to clarify this. Stress responses, physiological or cellular, influence energy use, cellular growth, and ultimately, health. A more complete understanding of the changes an animal undergoes during an immunological reaction may help us develop more effective future therapeutic applications and mitigate negative effects. However, before such experiments can be carried out and fully interpreted, basic research outlining responses of the animals in question to the process of infection must be conducted. 9 Thesis Objectives and Overview The primary objectives of this thesis were to investigate physiological processes underlying an acute Vibrio anguillarum infection and to provide new insights into responses brought about by an immune response, lethal or otherwise. An experimental approach was used in which studies were designed to examine responses to immunological challenge, both lethal and non-lethal, at both whole animal and cellular levels. While there are a great many methods available for assessing the state of stress an organism is undergoing, I have chosen for the purposes of this thesis to focus on measurement of plasma Cortisol and glucose as indicators of physiological stress. I have chosen to investigate the changes in hsp70 in two tissues where immunological agents are known to localize as a measurement of cellular stress, and to examine the effects of stress on non-specific immune function, I report changes in haematology and plasma lysozyme activity. Chapter One describes a study that was undertaken to investigate responses of juvenile salmonids to a sub-lethal environmental challenge. This work was a collaborative project designed to examine both how fish responded at a physiological level to an environmental toxicant at concentrations well below those allowed by government, and how they coped with a subsequent pathogenic challenge. The importance of this study lies in the current environmental standards that consider only standardized toxicity tests when setting guidelines. There are several inherent problems with the current guidelines because they are based on examination of short term survival, growth or reproduction. Most notably, they do not consider more subtle alterations in the ability of an animal to respond to subsequent challenge. Results from this study indicate that there are more subtle changes occurring at physiological and immunological levels and that current standards for environmental toxicants are not sufficiently conservative. Subsequent chapters shift to a closer examination of processes occurring during an immune response. Specifically, they were intended to examine stress effects of an immunological challenge. Chapter Two describes cellular and physiological stress responses during an acute disease process using V. anguillarum as a model pathogen. Clear responses were observed using both physiological and cellular stress indicators and the next logical step was to investigate responses during a non-lethal immunological challenge using both a killed bacterin based on the same pathogen, and lipopolysaccharide (LPS), the endotoxin common to Gram-negative bacteria. Chapter Three explores the possibility that responses are part of an immunological response rather than simply cellular damage. Results indicated that both LPS 10 and a bacterin were responded to on a physiological level as stressors. However, some differences between a pathogenic disease process and a non-lethal immunological challenge were striking. Cortisol increased during a live challenge and there is a known relationship between this stress hormone and hsp70. The study outlined in Chapter Four investigated this relationship during an active infection by blocking physiological effects of Cortisol through use of the antagonistic hormone analogue RU-486. While results from this study were somewhat less clear, what did become obvious was that the blocker may be an inappropriate compound for use in immunological studies and should be further investigated with respect to its effects in other physiological studies. The final component of this thesis, Chapter Five, reports effects of immunological challenge, using a virulent pathogen, a killed bacterial suspension, and LPS from two sources, at the cellular level. These experiments were carried out using cell cultures representing the two tissues examined throughout this thesis, liver and head kidney, and responses were examined both in the presence and absence of Cortisol. 11 References Ackerman, P. A. 1995. Effects of adjuvanted Aeromonas salmonicida vaccines on growth, oxygen consumption, and selected haematological variables in juvenile rainbow trout (Oncorhynchus mykiss). Department of Animal Science. Vancouver, BC, Canada, University of British Columbia: 94 p. Asea, A., S. K. Kraeft, E. A. Kurt-Jones, M. A. Stevenson, L. B. Chen, R. W. Finberg, G. C. Koo and S. K. Calderwood. 2000. HSP70 stimulates cytokine production through a CD14-dependent pathway, demonstrating its dual role as a chaperone and cytokine. Nature Medicine 6: 435-442. 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Jaquier-Sarlin, F. Sinclair and B. S. Polla. 1994. Exposure of monocytes to heat shock does not increase class II expression but modulates antigen-dependent T cell responses. International Immunity 6: 925-930. Maule, A. G., R. A. Tripp, S. L. Kaattari and C. B. Schreck. 1989. Stress alters immune function and disease resistance in chinook salmon (Oncorhynchus tshawytscha). Journal of Endocrinology 120: 135-142. 13 Mazeaud, M. M., F. Mazeaud and E. M. Donaldson. 1977. Primary and secondary effects of stress in fish: some new data with a general review. Transactions of the American Fisheries Society 106: 201-212. Mesa, M. G., T. P. Poe, A. G. Maule and C. B. Schreck. 1998. Vulnerability to predation and physiological stress responses in juvenile chinook salmon (Oncorhynchus tshawytscha) experimentally infected with Renibacterium salmoninarum. Canadian Journal of Fisheries and Aquatic Sciences 55: 1599-1606. Mestril, R., S. H. Chi, M. R. Sayen, K. O'Reilly and W. H. Dillman. 1994. Expression of inducible stress protein 70 in rat heart myogenic cell confers protection against simulated ischemia-induced injury. Journal of Clinical Investigation 93: 759-767. Mock, A. and G. Peters. 1990. Lysozyme activity in rainbow trout, Oncorhynchus mykiss (Walbaum), stressed by handling, transport and water pollution. Journal of Fish Biology 37: 873-885. Morimoto, R. I., A. Tissieres and C. Georgopoulos. 1990. The Stress Response, Function of the Proteins, and Perspectives. Stress Proteins in Biology and Medicine. R. I. Morimoto. Panjwani, N., L. Popova and P. K. Srivastava. 2002. Heat shock proteins gp96 and hsp70 activate the release of nitric oxide by APCs. Journal of Immunology 168: 2997-3003. Plytycz, B. and R. Seljelid. 1995. Nonself as a stressor - inflammation as a stress reaction. Immunology Today 16: 110-111. Rand, T. G. and D. K. Cone. 1990. Effects of Ichthyophonus hoferi on condition indices and blood chemistry of experimentally infected rainbow trout (Oncorhynchus mykiss). Journal of Wildlife Diseases 26: 323-328. Ritossa, F. 1962. A new puffing pattern induced by temperature shock and DNP in Drosophila. Experientia 18: 571-573. Robert, J., A. Menoret, S. Basu, N. Cohen and P. K. Srivastava. 2001. Phylogenetic conservation of the molecular and immunological properties of the chaperones gp96 and hsp70. European Journal of Immunology 31: 186-195. Robertson, L., P. Thomas, C. R. Arnold and J. M. Trant. 1987. Plasma Cortisol and secondary stress responses of red drum to handling, transport, rearing density, and a disease outbreak. Progressive Fish Culturist49: 1-12. Rokutan, K., T. Hirakawa, S. Teshima, Y. Nakano, M. Miyoshi, T. Kawai, E. Konda, H. Morinaga, T. Nikawa and K. Kishi. 1998. Implications of heat shock/stress proteins for medicine and disease. Journal of Medical Investigations 44: 137-147. Sanders, B. M. 1990. Stress Proteins: potential as multitiered biomarkers. Environmental Biomarkers. L. Shugart and J. McCarthy. Florida, Lewis Publishers: 165-191. Sanders, B. M. 1993. Stress proteins in aquatic organisms: an environmental perspective. Critical Reviews in Toxicology 23: 49-75. Schett, G., K. Redlich, Q. Xu, P. Bizan, M. Groger, M. Tohidast-Akrad, H. Kiener, J. Smolen and G. Steiner. 1998. Enhanced expression of heat shock protein 70 (hsp70) and heat shock factor 1 (HSF1) activation in rheumatoid arthritis synovial tissue. Journal of Clinical Investigation 102: 302-311. Selye, H. 1936. A syndrome produced by diverse nocuous agents. Nature 138: 32. 14 Selye, H. 1946. The general adaptation syndrome and the diseases of adaptation. Journal of Clinical Endocrinology 6: 117-230. Selye, H. 1950. Stress and the general adaptation syndrome. British Medical Journal 1950: 1383-1392. Wedemeyer, G. A., B. A. Barton and D. J. McLeay. 1990. Stress and Acclimation. Methods for fish biology. C. B. Schreck and P. B. Moyle. Bethesda, Maryland, American Fisheries Society: 451-489. Wedemeyer, G. A. and D. J. McLeay. 1981. Methods for Determining the Tolerance of Fishes to Environmental Stressors. Stress and Fish: 247-275. Wendelaar Bonga, S. E. 1997. The stress response in fish. Physiological Reviews 77: 591-625. Young, R. 1990. Stress proteins as immune targets in bacterial and parasite infections. Immune Recognition and Evasion. Molecular Aspects of Host-Parasite Interaction. L. H. T. Van Der Ploeg, R. C. Cantor and H. J. Vogel. San Diego, Academic Press: 315 pp. 15 Section I. Stress responses of juvenile salmonids to external and internal challenge. Environmental challenges come in many guises. Aquatic organisms are exposed to temperature fluctuations, pH shifts, changes in environmental gasses, pollutants and toxicants to name a few. Such environmental challenges can result in a number of changes that can be detrimental to health. While we tend to think of the environment in terms of being external to the body, the internal environment is tightly managed and many environmental challenges we face each day are internal threats. Common pathogens are encountered on a daily basis and the manner in which the body responds to them determines the outcome of these experiences. It is well documented that external environmental stressors are capable of eliciting physiological stress responses and many of these responses are able to influence the outcome of a subsequent pathogen encounter. Environmental standards are set on the basis of studies that assess survival, but questions have been raised that cast doubt on their capacity to provide sufficient protection for health of aquatic organisms and their ability to withstand a subsequent challenge such as that of a pathogen. The goals of the experiments described in this section were threefold. 1) To measure physiological responses of fish to the toxicant ammonia at well below environmental standards set by government. 2) To investigate the ability of fish to fight a subsequent infection with the pathogen Vibrio anguillarum. 3) To describe stress related changes within a fish during the course of infection. 16 CHAPTER 1 Effects of sub-lethal environmental ammonia exposure on recently saltwater acclimated juvenile Chinook salmon (Oncorhynchus tshawytscha) Introduction The chinook salmon, Oncorhynchus tshawytscha, is important as both a wild fishery and a cultured species. Wild chinook salmon spawn in streams and rivers that are tributaries to the Pacific Ocean. After spending their early life stages in freshwater they undergo smoltification allowing them to move into a marine environment following downstream migration. Very little is known about the initial marine phase of salmonids (Thorpe 1994); however there is a holding period in estuaries prior to fish moving out into the ocean (McCormick et al. 1998). Final stages of downstream migration are typically in the main stems of rivers and estuaries, areas that can have a high level of industry associated with them due to geographical distribution of large cities. One consequence of concentrated industry is water pollution, including ammonia (API 1981). In aquaculture, ammonia toxicity may also be problematic during freshwater rearing stages in which fish are held at high densities with minimal water flow, and as use of re-circulating water systems increases, resulting in increases in ammonia in the water. Ammonia is an unusual toxicant in that it is produced naturally as a metabolic waste product of amino acid catabolism. It is also released into the environment through production of industrial fertilizers and biological wastes. It is toxic to animals if accumulated in tissues, and fish rely mainly on diffusion down the concentration gradient between the body and water to eliminate it, although exact mechanisms are somewhat controversial (Evans and Cameron 1989). If the concentration in surrounding water exceeds that of blood, it becomes difficult to remove and conversion to non toxic forms becomes a priority (Vedel et al. 1998). Toxicity of ammonia on aquatic organisms has been well studied and results from toxicity tests have been used to set environmental protection standards (EPA 1984). In general its effects on fish are considered less severe in saltwater than in freshwater although most studies have taken place in freshwater and there is a relatively small saltwater data set available (Handy and Poxton 1993). Water pH in the marine environment tends to be higher than in many freshwater systems and this increased pH shifts ammonia equilibrium to favour a higher concentration of NH 3 , the lipophilic form of ammonia that can readily cross gill membranes (Evans 1989). The US EPA (1989) has established ammonia water quality criteria for saltwater, but these are based on a much smaller data set than freshwater standards. The 17 limited research that has been conducted on ammonia toxicity in fish in the marine environment rarely takes into consideration life history of test species. The nature of the salmonid life cycle can place young salmon in areas of potentially high ammonia concentrations at the end of the smoltification process and downstream migration. There have been several studies indicating that salmon smolts are more susceptible than other life-stages to anthropogenic toxicants; examples include mild acidity in combination with aluminum (Staurnes ef al. 1996), copper, and zinc (Lorz ef al. 1976). Since ammonia is toxic to fish it is possible that salmon in the smolt stage may be more susceptible to ammonia toxicity than other life stages because of changes they are undergoing. The marine environment also presents new challenges that juvenile fish must face, one of which is exposure to potential pathogens. Toxicants such as aromatic and chlorinated compounds in estuaries can compromise the immune system of juvenile chinook salmon, resulting in increased susceptibility to a disease such as vibriosis (Arkoosh ef al. 2001) a disease caused by a common marine bacterial pathogen, Vibrio anguillarum. Increased mortality rates associated with disease in toxicant exposed salmon are likely due to a compromised immune system. The combination of a low level of ammonia exposure, smoltification stress and disease challenge could pose major challenges for these fish. Current North American saltwater standards are based on a limited marine database and toxicity tests follow standard guidelines using static water conditions, unfed, unstressed, resting animals (ASTM 1993). It is under these unrealistic conditions that internal ammonia production is minimal. In addition, the available literature generally expresses concern primarily with growth, survival, and reproduction in fish. This study was designed to examine applicability of these standards to more realistic conditions (fed fish in culture situations and in nature), consider effects of a sub-lethal chronic ammonia exposure on recently saltwater acclimated chinook salmon and determine if a low grade ammonia exposure affected disease susceptibility in fish subsequently exposed to a V. anguillarum infection. Specifically, we asked: "Is the health status of animals negatively affected at below allowable levels of ammonia in the water?" and "Are current standards for allowable ammonia levels in aquatic environments conservative enough?" Materials and Methods Fish - Unvaccinated juvenile chinook salmon (approximately 20 g each) were transported from Big Tree Creek Hatchery (Campbell River, British Columbia) to the Bamfield Marine Station (Bamfield, British Columbia) in July of 2000. Fish were acclimated in outdoor 18 tanks in fresh water for one month. Fish were fed daily with a commercial food at a rate of 2% body weight/d during the acclimation period and 1% daily during experiments. Excess food was removed 20 min after feeding. Fish were acclimated to saltwater over 5 d by increasing the proportion of sea water added to fresh water by 20% daily. Fish were then divided into groups of 65 and transferred to one of 5 outdoor 1200 L saltwater tanks. Fish were acclimated to the new tanks for one week prior to experimental onset in September 2000. Seawater was pumped from Bamfield Inlet by the marine station and water quality parameters were measured as follows: water pH 7.8, salinity 31 g/kg, temperature 11-12.5°C and dissolved oxygen 85-94% saturation. Water flow to each tank was 2 L/min and was checked at least twice daily. Ammonia Exposure - To begin the experiment fish were fed at 8:00 am, and 1 h later the ambient ammonia concentration was increased by addition of a stock solution of NH 4CI. Nominal concentrations were estimated at 0 (control), 2.5 and 10.0 mg Total Ammonia Nitrogen (TAN)/L. Ammonia concentration was held constant by addition of a stock NH4CI solution to each test tank using a peristaltic pump (Masterflex). Water ammonia was measured daily using the indophenol blue method (Ivancic ef al. 1984). Two tanks of 65 fish were used for each ammonia concentrations and one was used as a control. Sampling - Six fish from each tank were terminally sampled by concussion at 6, 48, 96 and 244 h (8 fish) after onset of ammonia exposure, and weighed and measured. Blood was extracted via caudal puncture using a heparinized syringe. Blood smears were made for later analysis of differential cell counts. Aliquots of whole blood were taken for determination of haemoglobin, haematocrit, erythrocyte numbers, and respiratory burst activity. The remaining blood was immediately separated into plasma and red cell components by centrifugation at 7,600 x g for 3 minutes. Plasma was aliquotted and frozen on dry ice for later determination of Cortisol, glucose, lysozyme, plasma protein, and ammonia. Liver and head kidney tissues were dissected out for later measurement of heat shock protein 70 (hsp70). Remaining fish were challenged with V. anguillarum (serotype 02) to investigate alterations in disease susceptibility following ammonia exposure. Analytical Procedures - Plasma ammonia concentration for each sample was determined using a commercially available kit (Sigma, 171-UV). Plasma Cortisol was measured using a competitive binding ELISA assay (Neogen, Lexington, Kentucky, USA). Blood haemoglobin levels were measured as cyanmethaemoglobin with modified Drabkin's reagent and a standard containing human haemoglobin (Sigma Chemical Co. kit 525-A). Haematocrit (% red blood cells), erythrocyte numbers and differential leucocyte counts were determined 19 using the methodology described by Houston (1990). Leucocytes were categorized as lymphocytes, neutrophils, monocytes and thrombocytes based on morphological characteristics described by Yasutake and Wales (1983) and Houston (1990). Following measurement of haematocrit, capillary tubes were cut below the buffy layer and the plasma and white cell fraction were expelled into individual wells on glass slides to measure respiratory burst activity (Anderson et al. 1992). Slides were incubated in a moist chamber for 60 min after which they were washed gently with phosphate buffered saline (PBS) to remove cells that had not adhered to the glass. A solution (1 mg/mL) of nitro blue tetrazolium (NBT) in PBS was added to each well and cells were incubated for 60 min. Slides were examined under the microscope at 40X power and cells that appeared to have morphological characteristics of monocytes or neutrophils and had a blue halo were counted as NBT positive. At least 100 cells/well were counted and data are expressed as phagocyte respiratory burst activity, as percentage of NBT positive cells relative to the total number of cells counted. Data were collected for the NBT assay only at 6 h and 244 h post exposure. Plasma glucose was determined using a micro-modification of the Trinder (1969) glucose oxidase method (available from Sigma (315)). Plasma protein levels were measured using the bicinchoninic acid procedure (Smith et al. 1985) and values are reported as bovine serum albumin (BSA) equivalents. Plasma lysozyme activity was determined by a modification of Litwack's (1955) method (Maule et al. 1996). The method was modified for use on a microplate reader and used 10 pL of plasma (or hen egg white lysozyme (HEWL) standard ), and 250 pL of 0.025% w/v Micrococcus lysodeikticus suspension in 0.06 M phosphate buffer (pH 6.2). The decrease in optical density over 20 min of incubation at 25°C is reported here as ug/mL equivalent of HEWL activity. Preparation of tissues, and dilution and determination of hsp70 was carried out by ELISA according to Ackerman et al. (2001) as a modification of Forsyth et al. (1997). Briefly, liver and head kidney samples were diluted with 10 volumes ice-cold lysis buffer (50 mM Tris, 5 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 1 u.M Pepstatin A, 1 pM Leupeptin and 0.015 pM Aprotinin), sonicated on ice, vortexed, and placed back on ice. Aliquots (20 pL) of each sample were frozen immediately on dry ice and stored at - 80°C. For the ELISA, aliquots of lysates were re-dissolved with an equal volume of 0.1 N NaOH and diluted to 30 ug/mL in coating buffer (50 mM sodium carbonate-bicarbonate, pH 9.6). Wells of ELISA plates (Dynatec Laboratories, lmmulon-4 ©) were coated with 50 pL of diluted lysate overnight. Wells were then blocked with 2% w/v skim milk powder in Tris buffered saline containing Tween-20 (TBS-T20) (20 mM Tris, 500 mM NaCl, 0.05% Tween-20, pH 7.5), for 1 h. Wells were washed 3 x 20 with TBS-T20 after each subsequent assay step. After blocking, contents were discarded and the plates were incubated at room temperature (20°C) for 1 h with rabbit anti-hsp70 (StressGen SPA-758), 100ul_/well 1/2000 and 1 h with alkaline phosphatase labelled goat anti-rabbit (GAR-AP) (Gibco-BRL 9815SA) (100 uL/well, 1/1000). The plates were incubated at room temperature for 20 h with 100 uL/well of substrate (1 g disodium p-nitro-phenolphosphate/L, 10% w/v diethanolamine, pH 9.5). Absorbance was measured with a microplate reader (Molecular Devices THERMOmax™. Total protein bound to each well was assumed to be equal and representative of proteins present in the lysate. A standard curve was constructed by using a positive control lysate containing 6.9 x more hsp70 than a normal liver lysate. Positive control lysate and normal coho liver lysate were mixed to produce a series of standards containing 30 pg protein/mL in coating buffer of which between 0% and 80% was contributed by positive control lysate and the balance by normal liver lysate. The standard curve was linear over the standard range. A linear regression was used to estimate hsp70 concentration of each sample in relative units as a percentage of hsp70 content of positive-control. All samples were run in triplicate. Disease Challenge - The challenge method and V. anguillarum isolate are described in Ackerman et al. (2001). The concentration of the V. anguillarum suspension was estimated from absorbance measurements made at 540 nm (1 O D 5 4 0 estimated to contain 109 cells/mL). The suspension was diluted and each fish received a volume of 100 uL for a dose of 10 6 cfu/fish (actual dose 1.7 x 10 6 cfu/fish as determined by drop plate counts). Fish remaining after sampling were fin clipped for later identification and pooled into 2 tanks with clean water (no added ammonia) for disease challenge. Each tank contained 25 each of control, low, and high ammonia treated fish. Fish from pooled tanks were injected with bacterial suspension (100 uL/fish). Tanks were monitored twice daily and cumulative mortality data were collected for 14 days. The remaining 25 fish were clipped and moved to a third tank and received a saline injection to serve as assurance that mortalities were not occurring as a result of some outside factor. All mortalities were determined to be due to V. anguillarum infection upon re-isolation of the pathogen from kidney tissue. Statistical Analysis - Data were analysed using a factorial model where appropriate and are presented as means + 1 standard error (SE). The two factors were ammonia, which had 3 levels: 0 (control), 2.5 and 10.0 mg TAN/L), and time, with 4 levels (6, 48, 96 and 244 h). Two replicates were used for the 2.5 and the 10.0 mg/L TAN treatments and one for control. The two replicates were pooled for plasma ammonia, Cortisol, and glucose when no significant difference was found in plasma ammonia concentrations. Plasma Cortisol data were log 21 transformed to normalize them and reduce error variance heterogeneity. Lysozyme data were normalized using a square root transformation. Where a significant effect was noted (P<0.05) a Bonferroni's test was used to determine where differences occurred. No statistical differences were found in mortality between tanks and data were pooled for final Chi-squared analysis. Respiratory burst activity data were analyzed using a one-way ANOVA followed by a Student-Newman Keuls all pairwise multiple comparison test. Results Ammonia Concentration - Actual water ammonia concentrations averaged over the duration of the experiment were below detection level in control tanks, 2.6 ± 0.5 and 12.1 ± 0.5 mg TAN/L in the low and high concentration groups respectively. There was a significant interaction between exogenous ammonia concentration and time for plasma ammonia. Plasma ammonia was significantly elevated at 6 h in both treatment groups compared both to control and each other (Figure 1-1). The highest concentration at this sampling time was in those fish exposed to 10.0 mg TAN/L (12.2 ±0.9 pg/mL) followed by the 2.5 mg TAN/L group (6.132 ± 0.4) and controls (3.73 ±0.5). At both 48 and 96 h, levels increased significantly in the high ammonia treatment group: 10.57 ± 0.5 and 9.61 ± 0.5 mg TAN/L respectively. This trend changed at the final sample period when again there was a significant increase in plasma ammonia in both treatment groups compared both to controls and each other. Plasma Cortisol - There was a large amount of individual variation in plasma Cortisol levels in all groups including controls. A significant interaction was identified between time and ammonia concentration (P<0.001) for plasma Cortisol. The trend suggests that plasma Cortisol increases with ammonia exposure over time; however only exposure to 2.5 mg/L resulted in a significant Cortisol increase at 96 h (281.5 ± 76.6) (Figure 1-2). Although the trend was similar for fish exposed to 10 mg/L, the increase was not significant when compared to 6 h values (Table 1-1) or controls at 96 h. Plasma Glucose - There was a significant interaction between time and exogenous ammonia concentration with respect to plasma glucose concentration. No significant difference was noted in control fish with time (Table 1-2) and mean values ranged from 81.4 to 100.8 ±10.3 mg/dL. Fish exposed to 2.5 mg TAN/L showed a significant peak at 96 h with a value of 121.2 ± 7.3 mg/dL when compared to both controls and other sampling times within this treatment group. Plasma glucose levels in fish exposed to 10.0 mg TAN/L decreased with time and the 244 h sampled fish had significantly lower glucose levels at 59.7 ± 6.3 mg/dL than both 22 those sampled at 48 h ( 89.1 ±7.6 mg/dL) (Table 1-2) and both controls and 2.5 mg/L exposed fish at 244 h (Figure 1-3). Plasma Lysozyme Activity - There was a significant interaction between time and exogenous ammonia exposure. Activity increased significantly in both treatment groups compared to control fish. At 96 h post exposure, both treatment groups had significantly greater activity than controls (2.333 ± 0.420 pg/mL HEWL Eq) and fish exposed to 10.0 mg TAN/L (4.050 ± 0.293 pg/mL HEWL Eq) had significantly higher activity than did fish exposed to 2.5 mg TAN/L (3.183 ±0.199 pg/mL HEWL Eq) (Figure 1-4) By 244 h, fish exposed to 2.5 mg TAN/L (1.988 ±0.138 pg/mL HEWL Eq) had significantly lower activity levels than control fish (2.737 ± 0.314 pg HEWL Eq). Hsp 70 - Liver hsp70 levels at 48 h were significantly different between treatments (Figure 1-5). Fish exposed to 2.5 mg TAN/L (57.173 ± 6.491 relative units) had levels that were significantly greater than control fish (43.884 ±1.010 relative units) or fish exposed to 10.0 mg TAN/L (38.818 ± 3.129 relative units). Levels did not vary significantly over time for either controls or fish exposed to 10.0 mg TAN/L but there was a significant variation in hsp70 levels in liver over time for fish exposed to 2.5 mg TAN/L with a peak level occurring at 48 h (Table 1-2). There was a significant interaction between time and dose. Hsp70 levels in head kidney tissue showed no significant differences over time (P<0.05) (Figure 1-6). However, at 48 h, fish exposed to 10.0 mg TAN/L (45.497 ± 10.788 relative units) had significantly less hsp70 in this tissue than controls (61.603 ± 9.848 relative units) or the 2.5 mg TAN/L group (68.058 ± 10.788 relative units). Respiratory Burst Activity - Reduction of NBT was used as a measure of respiratory burst activity of adherent leucocytes at 6 h and 244 h. At 6 h both treatment groups (2.5 mg TAN/L mean 34.348 ± 4.667 positive cells; 10.0 mg TAN/L mean 42.399 ± 6.754 positive cells) had higher levels of NBT reduction than controls (mean 9.048 ±3.142 positive cells). By 244 h post exposure there were no differences between groups. (Figure 1-7, Table 1-1) Haematology - Changes in levels of circulating leucocytes relative to erythrocytes were observed at 96 h: fish exposed to 10.0 mg TAN/L (16.366 + 2.703 leucocytes/103 erythrocytes) had significantly fewer circulating leucocytes than control fish (29.468 ± 3.659 lymphocytes/10 3 erythrocytes). There was a significant interaction between time and exposure dose. No differences were found in numbers of total leucocytes/mm 3 at any time for any treatment (Table 1-2). The high exposure dose (Least Square Mean (LSM) 1824.096 ± 23 148.310 lymphocytes/mm 3) had a significant overall effect on the number of lymphocytes/mm 3 blood compared to controls (LSM 2513.194 ± 208.358 lymphocytes mm 3). The higher dose (LSM 13.6000 ± 1.107 lymphocytes/103 erythrocytes) resulted in a significantly lower number of lymphocytes relative to erythrocytes compared to controls (19.386 ± 1.482 lymphocytes/10 3 erythrocytes). At 96 h the high dose group (11.304 ± 2.262 lymphocytes/10 3 erythrocytes) had a significantly lower number of than did controls (24.542 ± 3.062). At 244 h, fish exposed to 2.5 mg TAN/L (20.136 ± 2.080 lymphocytes/10 3 erythrocytes) had significantly greater numbers of lymphocytes than the high dose group (11.241 ± 2.262 lymphocytes/10 3 erythrocytes) (Table 1-3). Controls (16.802 ± 2.652 lymphocytes/10 3 erythrocytes) did not differ significantly from either treatment. There was a statistical interaction between dose and time. Both treatments had significant effects on the proportion of lymphocytes present in circulation. There was no significant difference between controls (LSM 83.065 ± 2.883 %) and 2.5 mg TAN/L (LSM 83.008 ±2.133 %) but 10.0 mg TAN/L (LSM 70.066 ±2.153 %) differed from both overall and at two points in time. At 48 h fish exposed to 10.0 mg TAN/L (70.663 ± 4.604 %) had a significantly lower percentage of lymphocytes than fish exposed to 2.5 mg TAN/L (85.453 ± 1.752 %) (Table 1-3). At 244 h post exposure, fish exposed to 10.0 mg TAN/L (61.144 ± 7.590 %) had a significantly lower percentage of lymphocytes than both controls (84.871 ± 5. 263 %) and the low dose group (81.907 ±3.135 %). There was a significant difference between effects of 2.5 mg TAN/L (LSM 5.189 ± 1.292 %) and 10.0 mg TAN/L (LSM 10.837 ± 1.337 %) on percentage of circulating neutrophils, but there was no interaction between time and'dose. At 48 h control fish (2.623 ± 1.663 %) had a significantly lower proportion of neutrophils than did fish exposed to 10.0 mg TAN/L (15.198 ± 4.534 %) (Table 1-3). No differences were observed in the proportion of neutrophils to erythrocytes, or in numbers of neutrophils/mL. Numbers of circulating thrombocytes were significantly greater in the high dose group, 10.0 mg TAN/L (LSM 3.078 ± 0.446 thrombocytes/103 erythrocytes), compared to the control fish (LSM 1.469 ± 0.614 thrombocytes/103 erythrocytes) (Table 1-4). The high dose, 10.0 mg TAN/L (LSM 15.350 ± 1.782 %) , resulted in an overall significant increase in percentage of thrombocytes compared with controls (LSM 7.353 ± 2.386 %) or with the low dose group, 2.5 mg TAN/L (LSM10.661 ± 1.765 %). At 244 h, fish exposed to 10.0 mg TAN/L (17.871 ± 3.045 %) had a significantly higher percentage of thrombocytes than did controls (2.676 ± 1.845 %) 24 and, although not significant, fish exposed to the low dose, 2.5 mg TAN/L (12.643 ± 3.715 %), showed a trend toward an elevated level of thrombocytes (Table 1-4). Mortality - Following treatments, all fish were moved to clean water and challenged with the pathogen V. anguillarum. Prior to disease challenge there was no mortality in any tank. Following challenge, control fish had the least mortality at 87%. Fish exposed to 2.5 mg TAN/L had a total cumulative mortality of 92%. This was not statistically different from control fish. Exposure to 10.0 mg TAN/L for 244 h resulted in 100% cumulative mortality when pathogenically challenged. This was a statistically significant difference from control fish (Figure 1-8). 25 Figure 1-1. Plasma ammonia concentration in juvenile chinook smolts exposed to three levels of environmental ammonia as total ammonia nitrogen (TAN). Means are presented +/- SE. Superscripts denote statistical difference by two way ANOVA (P < 0.05) within each sampling period only. 26 Figure 1-2. Plasma Cortisol concentrations of juvenile chinook smolts exposed to three levels of environmental ammonia as total ammonia nitrogen (TAN). Means are presented ± SE. Superscripts denote statistical difference by two way ANOVA (P < 0.05) within each sampling period only. 27 Figure 1-3. Plasma glucose concentrations of juvenile chinook smolts exposed to three levels of environmental ammonia as total ammonia nitrogen (TAN). Means are presented ± SE. Superscripts denote statistical difference by two way ANOVA (P < 0.05) within each sampling period only. 28 Figure 1-4. Plasma lysozyme activity in juvenile chinook smolts exposed to three levels of environmental ammonia as total ammonia nitrogen (TAN). Means are presented ± SE. Superscripts denote statistical difference by two way ANOVA (P < 0.05) within each sampling period only. 29 90 -i 80 H 70 H 60 H ^ 5 0 H o CL co 4 0 30 20 H 10 O 0 mg TAN/L • 2.5 mg TAN/L V 10.0 mg TAN/L 48 96 244 Time (h) Figure 1-5. Liver hsp70 concentrations in juvenile chinook smolts exposed to three levels of environmental ammonia as total ammonia nitrogen (TAN). Means are presented ± SE. Asterisks denote statistical differences by two way ANOVA (P < 0.05) within each sampling period only. 30 140 120 100 80 60 40 20 O 0 mg TAN/L • 2.5 mg TAN/L V 10.0 mg TAN/L 48 96 244 Time (h) Figure 1-6. Head kidney hsp70 concentrations in juvenile chinook smolts exposed to three levels of environmental ammonia as total ammonia nitrogen (TAN). Means are presented ± SE. Asterisks denote statistical differences by two way ANOVA (P < 0.05) within each sampling period only. 31 Total ammonia nitrogen (TAN) Figure 1-7. Concentration of nitro blue tetrazolium (NBT) positive cells at 6 and 244 h during exposure to three levels of ammonia as total ammonia nitrogen (TAN). Means are presented ± SE. Asterisk denotes statistical difference within each sampling period only (P < 0.05). 32 O 0 mg TAN/L • 2.5 mg TAN/L V 10.0 mg TAN/L Time (d) Figure 1-8. 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O CN d +1 00 00 CD oo d +1 Is-CD CJ) 00 o +1 in CN CO CO d +1 co o d 0 o o o CO +1 o 00 00 Is-co Is-d CN +1 00 CO LO I d co oo I s -CN CO +1 CD CD in co I CN CO CN CN +1 CD CN CN CO 00 I s -CN CO +1 00 CO 00 CD CN CN +1 00 in N" CO oo Is-C\i oo +1 o in CO d CN co| E lo m oo oo CN +i CO CN O cn CN • E E "55 0 % o o cz o 37 Discussion Levels of aquatic ammonia reportedly safe according to EPA standards for marine fish led to an accumulation of ammonia in plasma that corresponded with external levels and resulted in a number of stress related responses. We observed significant effects on plasma Cortisol, glucose and lysozyme activity. More importantly, when fish were returned to clean water and challenged with V. anguillarum, control fish fared better and had a significantly greater survival rate than those exposed to currently acceptable levels of ammonia. Ammonia levels chosen for the present study (10.0 mg TAN/L and 2.5 mg TAN/L) were deliberately well below EPA standards in order to more closely examine effects of sub-lethal exposure on ammonia metabolism, physiological and cellular stress responses, and susceptibility to disease. Current environmental standards for ammonia are based on toxicity tests that involve unfed, unstressed fish in static water (ASTM 1993). In nature, salmonids exist in flowing water, must use energy to feed and compete for other resources, all of which result in varying degrees of physiological stress. Tests also consider commonly measured indices such as survival and growth but not more subtle influences on innate immunity that can potentially pose larger post exposure consequences. Stress is known to have a variety of modulatory effects on immune function, some are adaptive, and others are considered maladaptive. It is generally accepted that production of Cortisol is an adaptive response to stress, but this depends on perspective. From a physiological perspective, an increase in Cortisol in response to a stressor results in a mobilization of energy resources required to cope (reviewed by Wendelaar Bonga 1997). Munck et al (1984) suggest that the function of stress induced Cortisol increase serves to protect host tissues against normal defensive reactions activated by stress and that this is accomplished by preventing those reactions from exceeding themselves and threatening homeostasis. Stress related increases in metabolic activity also contribute to an increase in ammonia production (Mommsen et al. 1999) and, although it has been suggested that a stress response may increase ammonia toxicity in fish, it has been found that feeding may provide protection from internal ammonia toxicity rather than exacerbating it (Wicks ef al. 2002). In the current study, plasma Cortisol, glucose, and lysozyme levels in fish exposed to 2.5 mg TAN/L increased significantly, characteristic responses to stress indicating that levels of ammonia in both internal and external environments were being responded to at this point. Classical toxicity tests generally last 96 h and it was at this point that the above responses were observed. Other studies have shown no effects of ammonia on Cortisol levels, only minor effects on plasma glucose and sodium levels during a 30 d sub-lethal exposure (Fivelstad ef 38 al. 1995; Knoph et al. 1996), and have not been conclusive. In the current study, most blood parameters had returned to, or near control levels by 10 d post exposure. Notable exceptions were glucose levels in the high exposure group being significantly lower than in control and low exposure fish (Figure 1-1). This may suggest that metabolic reserves of these fish were depleted resulting in a greater mortality when subsequently challenged by infection. Lysozyme activity was significantly elevated at 96 h post exposure (Figure 1-4) for both treatments providing evidence that there was an immunologically relevant response occurring. Interestingly, activity in fish exposed to the low concentration was significantly lower than that in control fish at the end of the experiment indicating a potential immunosuppression in these fish. Lysozyme has been shown to increase within minutes following stress (Demers et al. 1997) and it has been suggested that Cortisol may attenuate its release from neutrophils and macrophages (Ellis 1981). Glucocorticoids can effect a reduction in circulating lymphocytes and monocytes, reduce antibody formation in salmon (Espelid et al. 1996) and correlate with changes in immunologically active enzymes such as lysozyme. While stress responses provide access to metabolic resources through gluconeogenesis, other systems are down-regulated by increased Cortisol secretion, the immune system is particularly affected by stress responses. Inflammation, an important response that allows the body to respond quickly to injury or bacterial recognition factors such as LPS, is dampened by glucocorticoids. It is not known if the high concentration would have led to a similar decrease had the experiment been carried out for a longer period. Although levels of TAN in the water in the present study were well below EPA standards, reduced numbers of circulating lymphocytes were observed in higher concentration exposed fish. Neutrophils and monocytes form part of the front line defences against pathogens and are a component of the innate immune system (reviewed in Dalmo et al. 1997). They typically infiltrate areas of inflammation and release destructive compounds such as reactive oxygen species to destroy pathogens. They are chemotactic and arise from the kidney and their contribution to initial defences against pathogenic invasion is great; low numbers can indicate that an animal has a reduced ability to deal with a pathogenic challenge. The transient changes in monocytes and neutrophils in the present study suggest that, in the early period of exposure, animals may be better able to deal with an immune challenge than at later stages although the higher concentration of thrombocytes suggests that there was an increase in clotting capacity. High levels of circulating thrombocytes are an indication of chronic stress (Espelid et al. 1996). The high level of neutrophil activity as indicated by NBT results at 6 h post exposure also suggests that fish were better prepared to meet an immune challenge early 39 on in exposure. One early immunological response to a stressor is a change in circulating blood cell proportions. Maule et al. (1989) showed a decrease in the ability of the anterior kidney to generate specific antibody producing cells 4 h after an acute physical stress. Interestingly, although there may be a decrease in these cells, several studies have shown increased circulating neutrophils following stress (Tomasso et al. 1983; Angelidis et al. 1987; Ellsaesser et al. 1987;) and an inhibition of neutrophil apoptosis with Cortisol application (Weyts et al. 1998a; Weyts et al. 1998b) suggesting that although certain components of the acquired immune system may be downregulated with stress, certain non-specific components are up-regulated. Because the concentrations of ammonia that fish were exposed to were so low, it was not expected that any changes in the levels of hsp70 would be found. For this reason, the significant alterations in tissue hsp70 at 48 h were surprising. Even more curious were the nature of the changes. The increased hsp70 in the liver tissue of fish exposed to the low concentration of ammonia at 48 h (Figure 1-5) was greater than both controls and high concentration exposed fish which were not significantly different from each other. Hsps are commonly measured as indicators of cellular stress, and tissue levels increase in response to cellular insults ranging from heat to toxicant exposure (Kothary et al. 1982; Lindquist 1988; Morimoto etal. 1990; Mestril etal. 1994; Flanagan etal. 1995; Ryan 1996; Iwama etal. 1998; Feder et al. 1999; Iwama ef al. 1999). They have been implicated in immune functions and have been associated with inflammation, as immune targets in viral, bacterial and parasitic infections, and have been implicated in self-nonself recognition (Young 1990; Srivastava et al. 1991; Kaufmann 1992; Mollenhauer ef al. 1992; Jaquier-Sarlin ef al. 1994; Multhoff 1996; Santoro 1996). The liver is a centre for detoxifying the blood and it would follow that the higher the concentration, the more active the cells in this respect, yet there was no change in measurable tissue level in fish exposed to high concentrations. This suggests that either low concentrations are more stressful at a cellular level in liver, or that turnover rate was greater in fish exposed to the higher of the two concentrations. The decrease in the measured level of hsp70 in head kidney cells (Figure 1-6) at this same time point is in stark contrast to what was observed in the liver. One possibility may be that the most metabolically active cells in the head kidney were expelled into circulation by tissue contraction and had not replenished at this point. Monocytes and neutrophils produce the greatest levels of hsp70 in the body (Bachelet ef al. 1998), and during a physiologically or immunologically stressful event, there is a lymphocytopenia that corresponds to a decrease in the numbers of lymphocytes in the spleen and kidney (Peters and Schwarzer 1985). Following handling stress and social stress, rainbow 40 trout show both a qualitative and a quantitative reduction in lymphocytes in the spleen and head kidney (Peters and Schwarzer 1985). It is possible that such changes could be responsible for the decrease in the hsp70 levels in the head kidney. Several authors have reported a decrease in hsp levels when Cortisol levels are increased (Ackerman et al. 2000; Basu et al. 2001; Boone et al. 2002) and this interaction may have had an effect on levels of hsp70 in the liver at 96 h post exposure. However, the changes were not reflected in head kidney tissue. Hsp70 levels in the head kidney had returned to levels equivalent to those of controls and low dose exposure and may lend credence to the idea that the pool of cells high in hsp70 had moved from the area into general circulation and, by 96 h, that pool was being replenished in this haematopoeic tissue. Hsp is known to provide protection to subsequent physiological insults and is implicated in immune protection (Kaufmann 1992; Mollenhauer etal. 1992; Jaquier-Sarlin etal. 1994; Guzhova etal. 1998; van Eden et al. 1998; Camins et al. 1999; Ratner 1999; Basu et al. 2000; Signgh-Jasuja et al. 2001). Perhaps there was an impairment of the hsp response due to ammonia level. While it appears that there was no increase in the level of hsp70 in the liver, this cannot be conclusively determined from the current data. Equally probable, the rate of production may have been equal to the rate of protein use which would result in the appearance of no net change. Similarly, in the head kidney, there may also have been an increased breakdown of protein resulting in the apparent decrease. When fish were transferred to clean water and infected by injection with V. anguillarum, control fish fared better and had a significantly greater survival rate than did those exposed to the higher concentration of ammonia. Lower concentrations of ammonia also tended to result in a greater susceptibility, although not significantly. What is significant is the fact that, at such low levels, there is an obvious immunological impairment. The EPA standards do not take more subtle immunological indicators of stress into consideration and place significant weight on immediate effects rather than long term health. An animal may be able to cope adequately with the single stress of a toxicant, but the reality is that organisms are rarely faced with single challenges. More commonly there are several environmental, physiological and biological trials being confronted at any one time. Differences in environmental temperature, dissolved agents, threats from predation and potential pathogens are generally all being faced concurrently. To draw conclusions about security levels for safe exposure to a single contaminant seems |ess than ideal. The significant differences in the ability of fish to combat an infective process' following a reportedly benign ammonia level serves to highlight this point. It is interesting however, that the group that had a lower 41 measurable level of hsp70 in both tissues at 96 h (although only significantly lower in head kidney) also had the greatest mortality when challenged with V. anguillarum following return to clean water after a10 d ammonia exposure. The data indicate significant detrimental effects of currently accepted levels of environmental ammonia on physiological stress responses and health parameters. They also indicate that at such levels there is an increase in disease susceptibility in fish. The data are complex, and indicate multiple effects on animals. These effects are more subtle than those currently used to evaluate environmental threats to fish. It is becoming increasingly clear that existing standards deserve revisiting and re-evaluation to make them more conservative and relevant to long term health of animals rather than short term survival. The changes that were observed led to an interest in the physiological processes of the infection itself and initiated the question: "Would hsp70 levels be affected by acute infection?" Although it may seem intuitive that an infection should be perceived as stressful on some level, there is scarce information regarding physiological effects of fish diseases and such knowledge is valuable to the field of health. 42 References Ackerman, P. A., R. B. Forsyth, C. F. Mazur and G. Iwama. 2000. 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Canadian Journal of Fisheries and Aquatic Sciences 53: 1695-1704. Thorpe, J. E. 1994. Salmonid fishes and the estuarine environment. Estuaries 17: 73-93. Trinder, P. 1969. Determination of glucose in blood using glucose oxidase with an alternative oxygen acceptor. Annals of Clinical Biochemistry 6: 24-27. van Eden, W., R. van der Zee, A. G. A. Paul, B. J. Prakken, U. Wendiing, S. M. Anderton and M. H. M. Wauben. 1998. Do heat shock proteins control the balance of T-cell regulation in inflammatory diseases? Immunology Today 19: 303-307. Vedel, N.E., B. Korsgaard and F.B. Jensen, 1998. Isolated and combined exposure to ammonia and nitrite in rainbow trout (Oncorhynchus mykiss): effects of electrolyte status, blood respiratory properties and brain glutamine/glutamate concentrations. Aquatic Toxicology. 41:325-342. Wendelaar Bonga, S. E. 1997. The stress response in fish. Physiological Reviews 77: 591-625. Weyts, F. A. A., G. Flik, J. H. W. M. Rombout and B. M. L. Verburg-van Kemenade. 1998a. Cortisol induces apoptosis in activated B cells, not in other lymphoid cells of the common carp, Cyprinus carpio L. Developmental and Comparative Immunology 22: 551-562. Weyts, F. A. A., G. Flik and B. M. L. Verburg-van Kemenade. 1998b. Cortisol inhibits apoptosis in carp neutrophilic granulocytes. Developmental and Comparative Immunology 22(5/6): 563-572. Wicks, B. J. and D. J. Randall. 2002. The effect of feeding and fasting on ammonia toxicity in juvenile rainbow trout, Oncorhynchus mykiss. Aquatic Toxicology 59: 71-82. Yasutake, W.T. and J.H. Wales. 1983. Microscopic anatomy of salmonids: an atlas. United States Department of the Interior. Fish and Wildlife Service. Resource Publication 150. 190 pp. Young, R. 1990. Stress proteins as immune targets in bacterial and parasite infections. Immune Recognition and Evasion. Molecular Aspects of Host-Parasite Interaction, (eds) L. H. T. Van Der Ploeg, R. C. Cantor and H. J. Vogel. San Diego, Academic Press: 315 pp. 46 CHAPTER 2 Physiological and cellular stress responses of juvenile rainbow trout (Oncorhynchus mykiss) to vibriosis 1 Introduction Wild and cultured fish are often exposed to a variety of stressful conditions, and many studies have demonstrated that such stressors can affect the health of fish through negative effects on the immune system (Maule ef al. 1989). There are many potential stressors in fish culture that may affect levels of circulating stress hormones and consequently influence the immune system (Iwama ef al. 1999). Pathogens are naturally occurring biological stressors that may represent one of the most significant challenges an organism may encounter. The relations between stress and disease resistance in fish have been extensively examined and increased susceptibility to disease during stress has been shown (Maule ef al. 1989; Mock ef al. 1990; Fevolden ef al. 1992); however, the mechanisms are poorly understood and few studies have described progression offish diseases from a physiological perspective. Heat shock proteins (hsp) are a class of highly conserved proteins that have received a great deal of attention since their discovery (reviewed in Welch 1993). They are classified based on approximate molecular weight (for example, hsp90, hsp70, hsp60, hsp30), and, of these, hsp70 is one of the best characterized with respect to cellular function. Heat shock proteins are induced by virtually all known stressors if sufficiently intense (Feder ef al. 1999) and this has led to the use of the term stress protein by many researchers to refer to these same proteins (i.e. hsp70 vs. SP70). While early studies of the hsp response centered on their increase relative to heat and chemical stressors, much of the present focus has shifted to the roles of hsp in health and disease. The inflammatory response elicits a strong hsp induction (reviewed in Jaquier-Sarlin ef al. 1994), and DeNagel ef al. (1993) have suggested that hsp could be a factor in the assembly of MHC-class II peptide complexes and in the subsequent presentation of antigen. Mariethoz et al. (1994) showed that members of the hsp70 family optimize antigen presentation and suggested that this contributes to an efficacious immune response. Hsps are also immunodominant antigens in a variety of pathological states, and immune recognition of pathogen and tumour related hsp have been implicated as serving as a Ackerman, P. A., and G.K. Iwama. 2001. Physiological and cellular stress responses of juvenile rainbow trout (Oncorhynchus mykiss) to vibriosis. Journal of Aquatic Animal Health. 13:173-180. 47 first line of defence for the host organism. Because of their immunogenicity they are being examined closely for potential use in vaccines (Mizzen 1998). Recently, Forsyth ef al. (1997) observed the induction of hsp70 in the inflamed tissues of coho salmon (Oncorhynchus kisutch) infected with chronic Renibacterium salmoninarum, the causative agent of bacterial kidney disease (BKD). They observed increased levels in liver and head kidney tissues only off ish with clinical signs of disease, and they postulated that the response also might be present in fish suffering from other diseases. Thus, we undertook the present study and examined the responses of rainbow trout (O. mykiss) to acute vibriosis. Vibriosis is a systemic bacterial infection caused by the small, Gram-negative, motile bacterium Vibrio anguillarum. It is a disease which generally occurs in the marine or estuarine environment but can be a health problem in freshwater. Clinical signs and pathology of the disease include skin ulcers or a septicaemia characterized by erythema, haemorrhages and anaemia; red, necrotic, boil-like lesions in the musculature; erythema of the bases of the fins and around the mouth; and absence of a leucocytic response (reviewed in Bullock 1987). During an acute outbreak, mortalities may reach 100% in 5-8 days. The objective of this study was to examine the effects of an ongoing V. anguillarum infection on cellular and physiological stress responses of juvenile rainbow trout (O. mykiss). The present study is the first to examine effects of acute disease on physiological and cellular stress responses in fish. Materials and Methods Fish and rearing conditions - Juvenile rainbow trout (mean weight 53.4g ± 1.7g) were obtained from Colebrook Trout Farm, British Columbia (B.C.), Canada and maintained at the University of British Columbia South Campus Aquaculture Unit. Fish were randomly divided into twelve 80 L tanks (2 L/min water flow rate), to a final number of 25 fish per tank (final density approximately 17 g/L), and were acclimated to the water system for two weeks. Fish were fed daily to satiation. The water temperature then was gradually increased at a rate of 1°C per day from ambient 5°C to 12°C. Fish were held at 12°C for 14 d prior to the start of the experiment. Prior to the disease challenge, 10 fish were randomly sampled from the tanks (2 fish each per treatment and control tank) to establish baseline values for measured variables (see below). Fish had been reared in fresh water, had received no immunizations and were naive to V. anguillarum as measured by antibody titration. Disease Challenge - The primary isolate (Pacific Biological Station, Nanaimo, B.C., isolate number 98055) of V. anguillarum (serotype 02) was obtained from wild chum salmon 48 (O. keta) that had died from natural exposure to the pathogen. Twenty hour V. anguillarum cells were harvested from tryptic soy agar (TSA, supplemented with 1.5% NaCI) plates, transferred to sterile peptone-saline (P-S: 0.2 % peptone, 1.5% NaCI) and vortexed to make a suspension. The concentration of the V. anguillarum suspension was estimated from absorbance measurements made at 540 nm (1 O D 5 4 0 estimated to contain 109 cells/mL). The suspension was diluted to 10 6 cfu/mL and each fish received a 100 uL intraperitoneal injection of this suspension for a dose of 10 5 cfu/fish (9.196 x 10 4 cells/fish as determined by drop plate counts). Following light anaesthesia (50 mg/L MS-222 buffered with 50 mg/L N a H C 0 3 ; Syndel Laboratories, Vancouver, B.C.) fish from 6 tanks were injected with the bacterial suspension (100 uL/fish); fish from the control tanks received the peptone-saline carrier only (100 uL/fish). Sampling - Five test tanks and five control tanks were used for daily sampling of fish and the remaining two tanks were used for mortality data. Three fish were removed from two of the treatment and control tanks every 24 h and placed in a lethal dose of anaesthetic (500 mg/L MS-222 buffered with 500 mg/L NaHC0 3 ; Syndel Laboratories, Vancouver, B.C.). In order to mitigate the stress effects of netting, fish were never removed from the same tanks on consecutive days. Blood was collected from the caudal vessel with sterile, heparinized syringes for determination of viable pathogen counts, haematocrit and haemoglobin. Remaining blood was centrifuged (2000 x g for 5 min) and the plasma was removed, frozen on dry ice and stored at - 70°C for later analyses of protein, Cortisol, glucose, lysozyme, and ion concentrations. Liver and head kidney tissues were quickly dissected out, frozen immediately on dry ice, and stored at - 70°C for later analysis of hsp70. Analytical Procedures - Blood haemoglobin levels were measured as cyanmethaemoglobin with modified Drabkin's reagent and a standard containing human haemoglobin (Sigma Chemical Co. kit 525-A). Haematocrit (% red blood cells) was determined using the method described by Houston (1990). Viable pathogen numbers were determined according to the method described by Balfry et al. (1997). Plasma chloride concentrations [Cl "] were determined by coulometric titration (Haake Buchler Instruments digital chloridometer), and plasma calcium and magnesium concentrations ([Ca+] and [Mg 2 +]) were measured colorimetrically (Sigma Chemical Co. kit 587 and kit 595). Plasma glucose levels were determined using a microtized modification of the Trinder (1969) glucose oxidase method (Sigma Chemical Co. kit 315) to use smaller sample volumes, while plasma protein levels were determined using the bicinchoninic acid procedure (Smith et al. 1985) and values are reported as bovine serum albumin (BSA) equivalents. Plasma Cortisol concentrations were determined using a commercially available enzyme-linked immunosorbent assay (ELISA) kit 49 (Neogen Corp. Lexington, KY). The method was validated for use with rainbow trout plasma by running identical test samples by ELISA and radio-immunoassay (RIA) (GammaCoat, Incstar Corporation, Stillwater, Minn.). The RIA method has been used extensively for salmonid measurements and its specificity is known. Lysozyme activity in plasma was determined by a modification of Litwack's (1955) method (Maule et al. 1996). The method was modified for use on a microplate reader and used 15 pL of plasma (or hen egg white lysozyme (HEWL) standard), and 250 pL of 0.025% w/v Micrococcus lysodeikticus suspension in 0.06 M phosphate buffer (pH 6.2). The decrease in optical density over 20 min of incubation at 25°C is reported here as pg/mL equivalent of HEWL. Liver and head kidney samples were diluted with 10 volumes ice-cold lysis buffer (50 mM Tris, 5 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 1 pM Pepstatin A, 1 pM Leupeptin and 0.015 pM Aprotinin), sonicated on ice, vortexed, and placed back on ice. Aliquots (10 pL) of each sample were frozen immediately on dry ice and stored at -70°C. Preparation of tissues, and dilution and determination of hsp70 was carried out by ELISA according to Forsyth et al. (1997) and reported as units relative to a positive arsenite induced control. The hsp70 antibody used was prepared as outlined in Forsyth et al. (1997). The two tanks from which sampling did not occur were monitored daily for mortalities for three weeks following injection of fish with V. anguillarum. Fish that died were examined for the presence of V. anguillarum by streaking aseptically removed kidney swabs onto TSA supplemented with 1.5% NaCI. Creamy white colonies containing bacteria that Gram-stained negative and displayed motility under microscopic inspection were identified as V. anguillarum. Statistical Analysis - Data are presented as means ± 1 standard error (SE). When analysis of variance indicated significant differences and data passed the normality test, the Student-Newman-Keuls all pairwise multiple comparison test was used to identify significantly different means (P < 0.05). Where data failed the normality test, Dunn's method of all pairwise multiple comparison was used for this purpose. The level of significance in all tests was P < 0.05. Results and Discussion Vibriosis developed in infected fish in a manner similar to that reported for intraperitoneally injected coho salmon (Balfry 1996). The first mortality in the exposed group occurred 6 d following injection, and cumulative mortality reached a maximum of 84% at 8 d post-infection (Figure 3-1). No mortality or morbidity was observed in peptone-saline injected 50 controls. The viable counts of V. anguillarum in the blood increased significantly 3 d post infection until day 6 at which time counts increased more than 20 fold in the blood (Figure 3-1). Fish that were sampled at 7 d and 8 d post-challenge still had significantly higher V. anguillarum counts than controls, but the level had begun to decline, suggesting that these surviving fish had successfully combated bacterial infection. In the present study we observed fluctuating haemoglobin and haematocrit levels in fish injected with V. anguillarum. Haemoglobin levels (g/dL) ranged from 4.25 ± 0.46 to 8.46 + 1.25 in control fish and from 2.85 ± 0.76 to 7.31 ± 0.41 in challenged fish. Significant differences (n=6, P=0.002) in haemoglobin between challenged and control fish were observed only at day 6 (controls: 5.78 ± 0.71, challenged: 2.847 ± 0.76). Three of the six challenged fish sampled on day 6 displayed clinical signs of disease. Haematocrit levels (% RBC) ranged from 29.5 ± 2.36 to 40.0 ± 2.03 in control fish and from 24.0 ± 3.24 to 43.0 + 5.98 in challenged fish. Haematocrit was consistently lower in infected fish compared to controls, although differences were not significant, except for in fish sampled 8 d post-challenge. At that time challenged fish had significantly (n=6, P=0.001) lower haematocrit levels (43.0 ± 5.98) compared to control fish (29.5 ± 2.36). Changes in the blood of fish infected with R. salmoninarum usually include anaemia, depressed plasma protein and glucose levels, and compromised ionic regulation (Iwama et al. 1986), but physiological data relating to V. anguillarum were not available to date. The significant decrease in haemoglobin seen in infected fish in the present study may have been a result of tissue damage in the kidney that would impair osmoregulatory processes (Hickman et al. 1969) and result in haemodilution for fish in fresh water. There was a significant difference in plasma Cortisol levels between control and infected fish 5 d following challenge. Cortisol levels remained significantly elevated for the duration of the experiment (8 d) although levels were on a decreasing trend after 5 d post-infection (Figure 3-2). Stress is known to elicit a variety of physiological responses that aid an organism in maintaining homeostasis in the face of internal or external perturbations. The increase in plasma Cortisol concentration in response to acute stress is one such example. There are very few reports of the effects of disease progression on plasma Cortisol concentration in fish (Suzumoto etal. 1977; Robertson etal. 1987; Laidley etal. 1988; Rand et al. 1990; Mesa et al. 1998) and available data are often conflicting, depending on the pathogen studied. Mesa et al. (1998) reported an increase in plasma Cortisol concentrations during progression of BKD in chinook salmon (O. tshawytscha) but changes occurred during later stages of infection. However, Suzumoto ef al. (1977) reported a decrease in Cortisol during 51 development of BKD. Robertson et al. (1987) reported a progressively increasing plasma Cortisol concentration in red drum (Sciaenops occelatus) in response to a natural infection by an unidentified bacterial pathogen. Laidley et al. (1988) described a lack of significant Cortisol increase in rainbow trout infected with the haemoflaggelate Cryptobia salmositica, and Rand et al. (1990) also saw a lack of a Cortisol response to infection with the fungus Ichthyophonus hoferi. As a result of their findings, Rand et al. (1990) suggested that activation of the hypothalamic-pituitary interrenal axis and secretion of Cortisol might not be a normal component of the stress response of fish to disease. It is difficult to reconcile these differences without accompanying physiological data to better understand the state of the fish, but conflicting data on Cortisol and disease in fish underscores the need for more comprehensive descriptions of pathophysiology of fish diseases. The present data support the studies that show a significant increase in plasma Cortisol in response to bacterial pathogens. Chronic Cortisol elevation is considered to have negative effects' on disease resistance (Maule ef al. 1989), and interferes with lymphocyte function (Tripp ef al. 1987; Espelid ef al. 1996). The glucocorticoids have a variety of inhibitory effects on immunity due to actions on the cells of the immune system, and Cortisol has been shown to interfere with lymphocytes by inhibiting their transformation into antibody producing cells and by inhibiting the release or synthesis of cytokines required to stimulate antibody production (reviewed in Colombo ef al. 1989; Marx 1995). It is possible that the elevated Cortisol over time was a contributing factor to the deterioration of those fish succumbing to vibriosis. However, more recent studies have indicated that Cortisol may have immunologically protective effects. It has been shown to reduce apoptosis in neutrophils (Weyts et al. 1998) suggesting that an increased level of Cortisol during times of stress may be beneficial to innate immune function by enhancing phagocytic defences and increasing chances of survival. In contrast to lower plasma glucose levels associated with chronic infection (Iwama ef al. 1986; Rand ef al. 1990; Mesa ef al. 1998), we saw no significant differences in plasma glucose levels during this acute vibrio challenge. Plasma protein and ion levels did not differ between controls and infected fish until 6 d post-challenge. At this point infected fish had significantly lower plasma protein concentration (challenged fish: 9.22 ± 2.00 mg/ml_, control fish: 23.46 ± 2.89 mg/mL) and plasma [CI-] (challenged fish: 111.58 ± 3.71 mmol/L, control fish 130.83 + 2.06 mmol/L) supporting our conclusion that fish at 6 d post-challenge were experiencing haemodilution due to kidney damage and impairment of osmoregulatory ability. However, no changes were observed in plasma [Ca+] or [Mg 2 + ] . Such probable haemodilution would have contributed to a masking of any increase in concentration of any of the measured 52 variables. Thus, plasma glucose, [Ca+] or [Mg2+] may have been increasing at a rate similar to the degree of haemodilution. Levels of hsp70 were measured in head kidney and liver tissues. These organs were chosen as sites where the pathogen is known to accumulate and were tissues that would likely show changes in hsp70 levels as a result. The antibody used in the ELISA has been shown to not cross react with bacterial antigen in the 70 kD range (Forsyth ef al. 1997); the observed hsp70 was therefore assumed to be from the fish tissue. Levels of hsp70 were significantly greater in head kidney tissue of infected fish than control fish by 4 d post-challenge, and increased over control levels in liver tissue of infected fish by 5 d post-challenge. Liver hsp70 reached a peak level at 5 d and was successively lower in fish over the remaining sampling periods, returning to control levels by 7 d post-challenge (Figure 3-3). Head kidney hsp70 in infected fish reached a maximum level at 6 d post-challenge, and returned to control levels by 8 d (Figure 3-4). In both tissues, levels of hsp70 were significantly increased prior to the highest level of pathogen in the blood and prior to any clinical signs of disease. The increase in hsp70 in V. anguillarum-'mfecied fish may represent a physiological reaction to tissue damage or physiological perturbations as a result of disease progression. The different patterns in the two tissues may represent localization of the pathogen or degree of tissue damage. Although significant differences were seen between infected and control fish, in both tissues, they were not as marked as those seen by Forsyth ef al. (1997). These authors suggested that the apparent increase in hsp70 actually may be a result of the degradation of proteins other than hsp70 rather than an actual induction of hsp70. We suggest that there may be other underlying reasons for the increase as there are some data to support a hypothesis that the increase in hsp70 during an acute infection such as vibriosis may be part of an immune response (reviewed in Jaquier-Sarlin ef al. 1994; Bachelet ef al. 1998). This clearly warrants further study into the cause and functions of the changes in hsp70 levels during the disease process. Plasma lysozyme activity (pg/mL HEWL Eq) ranged from 8.255 + 1.915 to 16.93 ± 1.38 for control fish and from 7.91 ± 1.24 to 16.07 ± 1.69 for challenged fish. Lysozyme activity fluctuated over time in both treatment and control fish which is likely attributable to variation within the species (Grinde ef al. 1988). Significant differences (n = 6, P < 0.001) between control fish and challenged fish were observed only at day 6 (control fish: 12.82 ± 0.85, challenged fish: 15.92 + 0.38). Lysozyme is an important plasma protein that has antimicrobial actions against Gram-positive bacteria (Grinde 1989; Lie ef al. 1989). Plasma lysozyme levels have been shown to increase within minutes following stress (Demers ef al. 53 1997) in rainbow trout. The highest concentrations of plasma lysozyme in the present study occurred when the total plasma protein concentrations were at their lowest levels. This was also the period when liver and head kidney tissue hsp70 was at maximal observed levels. Certain substances such as LPS and glucocorticoids can induce increased secretion of lysozyme (Altschemied et al. 1989). In fish however, Cortisol has been reported to have an attenuating effect on the release of lysozyme from neutrophils and macrophages (Ellis 1981). There was no significant change in lysozyme activity in the present study except for 6 d post-challenge when plasma levels were significantly higher than control values. This may have been due to the effects of Cortisol or, alternatively, the increase in lysozyme may not have occurred until a critical number of bacteria were reached in the blood and other defences had been overwhelmed. Given the possible attenuating effects of Cortisol and haemodilution, there may have been an actual stimulation of lysozyme production in the latter stages of the disease that may have been masked. Phagocytes represent an important component of the first line of defence against many pathogens (Cohen 1991). In mammals, hsp70 is found at higher levels in the monocyte/macrophage line of cells than in any other cell type tested and functions ranging from self/non-self recognition to antigen presentation have been attributed to this family of proteins (reviewed in Bachelet et al. 1998). Bacterial infection frequently results in activation of immunocytes and an increase in phagocytic killing activity through mechanisms such as the production of lysozyme, reactive oxygen species (i.e. hydrogen peroxide, hydroxyl radical) brought about by respiratory burst activity, and cationic proteins. These molecules are also damaging to components of host cells and it has been proposed that one function of hsp production in these cells is to protect against apoptosis due to auto-oxidation (Jaquier-Sarlin et al. 1994). The increase in hsp70 level just prior to the onset of clinical signs of vibriosis and mortalities suggests that hsp synthesis was occurring in the early stages of the response to infection and that the induction of the cellular stress response may be an important component in the early pathology of acute bacterial diseases. Alternatively, this may represent a response to physiological stress brought on by increasing pathogen load in the fish. Further studies are required to clarify the role that hsp70 plays in the pathophysiology of vibriosis and other fish diseases. Such investigations will likely shed more light on interactions between host and pathogens at the cellular level. 54 100 90 rtal 80 o 70 E ive 60 CO 50 E 40 ZJ o 30 1 * c 20 Q) O i _ CD 10 Q _ O control A infected Q V. anguillarum 0 1 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 1920 T i m e (d) Figure 2-1. Mean (+ SE) viable V. anguillarum in colony forming units per mL of blood and cumulative percent mortality for juvenile rainbow trout challenged with V. anguillarum by intraperitoneal injection (9.196 x 10 4 cells/fish). Significant differences in blood pathogen counts between days are noted by different letters (P < 0.001). 55 Figure 2-2. Viable V. anguillarum in blood and plasma Cortisol concentration following injection challenge into juvenile rainbow trout. Means are presented + SE. Significant differences in blood pathogen counts between days are indicated by different letters (P < 0 .001). Significant plasma Cortisol level differences between control and infected fish are noted with asterisks (P < 0.001). 56 Figure 2-3. Hsp70 levels in liver of juvenile rainbow trout experimentally infected with V. anguillarum compared with sham injected controls. Means are presented ± SE. Significant differences between control and infected measures are denoted by asterisks (P < 0.0001). 57 22 2 0 H O controls A infected § 18 jo 16 CD o 14 12 10 8 0 1 3 4 5 T i m e (d) 8 Figure 2-4. Mean (± SE) hsp70 levels in liver of juvenile rainbow trout experimentally infected with V. anguillarum compared with sham-injected controls. 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I s -T _ 00 co o LU O LU o co O 61 References Bachelet, M., C. Adrie and B. S. Polla. 1998. Macrophages and heat shock proteins. Research in Immunology 149: 727-732. Balfry, S. K., and G.K. Iwama. 1996. Non-specific factors important to innate disease resistance in coho salmon from British Columbia. Bulletin of the Aquaculture Association of Canada March 1996. no. 96-1: 20-22. Balfry, S. K., M. Shariff and G. K. Iwama. 1997. Strain differences in non-specific immunity of tilapia Oreochromis niloticus following disease challenge with Vibrio parahaemolyticus. Diseases of Aquatic Organisms 30: 77-80. Bullock, G. L. 1987. Vibriosis in fish. Washington, D.C., United States Department of the Interior, U.S. Fish and Wildlife Service. Cohen, M. S. 1991. Phagocytes in health and disease. Current Opinions in Infectious Disease 4: 338-343. Colombo, L, A. D. Pickering, P. Belvedere and C. B. Schreck. 1989. Stress inducing factors and stress reaction in aquaculture. Aquaculture Europe '89 - Business Joins Science, Bordeaux, France, European Aquaculture Society. Demers, N. E. and C. J. Bayne. 1997. The immediate effect of stress on hormones and plasma lysozyme in rainbow trout. Developmental and Comparative Immunology 21: 363-373. DeNagel, D. C. and S. K. Pierce. 1993. Heat shock proteins in immune responses. Critical Reviews in Immunology 13: 71-81. Ellis, A. E. 1981. Stress and the modulation of defence mechanisms in fish. Stress and Fish. A. D. Pickering. London, Academic Press, pp. 147-169. Espelid, S., G. B. Lokken, K. Steiro and J. Bogwald. 1996. Effects of Cortisol and stress on the immune system in Atlantic salmon (Salmo salar L.). Fish and Shellfish Immunology 6: 95-110. Feder, M. E. and G. E. Hofmann. 1999. Heat shock proteins, molecular chaperones, and the stress response: Evolutionary and ecological physiology. Annual Review of Physiology 61: 243-282. Fevolden, S. E., T. Refstie and K. H. Roed. 1992. Disease resistance in rainbow trout (Oncorhynchus mykiss) selected for stress response. Aquaculture 104: 19-29. Forsyth, R. B., E. P. M. Candido, S. L. Babich and G. K. Iwama. 1997. Stress protein expression in coho salmon with Bacterial Kidney Disease. Journal of Aquatic Animal Health 9: 18-25. Grinde, B. 1989. Lysozyme from rainbow trout, Salmo gairdneri Richardson, as an antibacterial agent against fish pathogens. Journal of Fish Diseases 12: 95-104. Hickman, C. P. and B. F. Trump. 1969. The kidney. Fish Physiology. W. S. Hoar and D. J. Randall. New York, Academic Press. 1: 9-239. Houston, A. H. 1990. Blood and circulation. Methods for fish biology. C. B. Schreck and P. B. Moyle. Bethesda, Maryland, American Fisheries Society: 273-334. Iwama, G. K., G. L. Greer and D. J. Randall. 1986. Changes in selected hematological parameters in juvenile chinook salmon subjected to a bacterial challenge and a toxicant. Journal of Fish Biology 28: 563-572. 62 Iwama, G. K., M. M. Vijayan, R. B. Forsyth and P. A. Ackerman. 1999. Heat shock proteins and physiological stress in fish. American Zoologist 39: 901-909. Jaquier-Sarlin, M. R., K. Fuller, A. T. Dinh-Xuan, M. J. Richard and B. S. Polla. 1994. Protective effects of hsp70 in inflammation. Experientia 50: 1031-1038. Laidley, C. W., P. T. K. Woo and J. F. Leatherland. 1988. The stress response of rainbow trout to experimental infection with the blood parasite Cryptobia salmositica Katz, 1951. Journal of Fish Biology 32: 253-261. Lie, O., O. Evensen, A. Sorensen and E. Froysadal. 1989. Study on lysozyme activity in some fish species. Diseases of Aquatic Organisms 5: 1-5. Litwack, G. 1955. Photometric determination of lysozyme activity. Proceedings for the Society for Experimental Biology and Medicine 89: 401-403. Mariethoz, E., F. Tacchini-Cottier, M. R. Jaquier-Sarlin, F. Sinclair and B. S. Polla. 1994. Exposure of monocytes to heat shock does not increase class II expression but modulates antigen-dependent T cell responses. International Immunity 6: 925-930. Marx, J. 1995. How the glucocorticoids suppress immunity. Science 270: 232-233. Maule, A. G., R. Schrock, C. Slater, M. S. Fitzpatrick and C. B. Schreck. 1996. Immune and endocrine responses of adult chinook salmon during freshwater immigration and sexual maturation. Fish and Shellfish Immunology 6: 221-233. Maule, A. G., R. A. Tripp, S. L. Kaattari and C. B. Schreck. 1989. Stress alters immune function and disease resistance in chinook salmon (Oncorhynchus tshawytscha). Journal of Endocrinology 120: 135-142. Mesa, M. G., T. P. Poe, A. G. Maule and C. B. Schreck. 1998. Vulnerability to predation and physiological stress responses in juvenile chinook salmon (Oncorhynchus tshawytscha) experimentally infected Renibacterium salmoninarum. Canadian Journal of Fisheries and Aquatic Sciences 55: 1599-1606. Mizzen, L. 1998. Immune responses to stress proteins: Applications to infectious disease and cancer. Biotherapy 10: 173-189. Mock, A. and G. Peters. 1990. Lysozyme activity in rainbow trout, Oncorhynchus mykiss (Walbaum), stressed by handling, transport and water pollution. Journal of Fish Biology 37: 873-885. Rand, T. G. and D. K. Cone. 1990. Effects of Ichthyophonus hoferi on condition indices and blood chemistry of experimentally infected rainbow trout (Oncorhynchus mykiss). Journal of Wildlife Diseases 26: 323-328. Robertson, L., P. Thomas, C. R. Arnold and J. M. Trant. 1987. Plasma Cortisol and secondary stress responses of red drum to handling, transport, rearing density, and a disease outbreak. Progressive Fish Culturist 49: 1-12. Smith, P. K., R. I. Krohn, G. T. Hermanson, A. K. Mallia, F. H. Gartenr, M. D. Provenzano, E. K. Fujimoto, N. M. Goeke, B. J. Olson and D. C. Klenk. 1985. Measurement of protein using bicinchoninic acid. Analytical Biochemistry 150: 76-85. Suzumoto, B. K., C. B. Schreck and J. D. Mclntyre. 1977. Relative resistances of three transferrin genotypes of coho salmon (Oncorhynchus kisutch) and their hematological responses to bacterial kidney disease. Journal of the Fisheries Research Board of Canada 34: 1-8. 63 Trinder, P. 1969. Determination of glucose in blood using glucose oxidase with an alternative oxygen acceptor. Annals of Clinical Biochemistry 6: 24-27. Tripp, R. A., A. G. Maule, C. B. Schreck and S. L. Kaattari. 1987. Cortisol mediated suppression of salmonid lymphocyte responses in vitro. Developmental and Comparative Immunology 11(3): 565-576. Welch, W. J. 1993. How cells respond to stress. Scientific American 5: 56-64. Weyts, F. A. A., G. Flik and B. M. L. Verburg-van Kemenade. 1998. Cortisol inhibits apoptosis in carp neutrophilic granulocytes. Developmental and Comparative Immunology 22: 563-572. 64 Section II. The effect of non-lethal immune challenge on stress responses While the live challenge study did describe changes in Cortisol and hsp70 during progression of vibriosis that have not been described previously, it did not address whether responses were solely due to the presence and possible actions of the live pathogen or to its cellular components. If the response is to the presence of bacterial cells themselves, then use of killed bacteria should result in a response similar to that seen using a live challenge. Bacterial LPS injected into fish as an adjuvant in conjunction with a bacterin has been suggested as a means to boost the natural immune system and enhance acquired immunity to the immunogen. Immunizations such as these are often utilized and an investigation into physiological effects of such immunizations has the potential to assist developers in formulating more efficacious vaccines that have fewer deleterious effects. Responses to LPS for hsp70 induction may also provide valuable insights into mechanisms of immunostimulation by LPS in fish and provide supporting evidence for the hypothesis that increased levels of hsp70 may be part of the immune response to bacterial disease. A further study was undertaken to investigate if and how fish responded at cellular and physiological levels to a non-lethal immunological challenge. 65 CHAPTER 3 Physiological and cellular stress responses of juvenile rainbow trout (Oncorhynchus mykiss) to LPS and a Vibrio anguillarum bacterin. Introduct ion Infection is a common biological threat and bacterial infection in fish has been shown to induce the hsp70 response. In a chronic Renibacterium salmoninarum infection (Forsyth et al. 1997) levels of hsp70 are not seen to increase until very late stages of disease and only in fish with severe clinical signs of disease. However, this pathogen has evolved a novel strategy for survival within the host and is extremely efficient at avoiding the host immune system; it resides within leucocytes and does not produce highly toxic compounds. As such, the host immune system is not particularly reactive to it. In an acute infection with Vibrio anguillarum, Ackerman et al. (2001) showed that an increase of hsp70 preceded any clinical signs of infection (or onset of mortalities) and occurred concurrently with a significant increase in plasma Cortisol. Vibrio anguillarum, a Gram-negative bacterium, often causes severe lesions and a general septicaemia and is considered a much more aggressive pathogen than R. salmoninarum. All Gram-negative bacteria produce compounds known as endotoxins. One of the most widely studied of these is lipopolysaccharide (LPS) which is particularly toxic to most homeotherms causing shock, haemorrhage, fever and death. Interestingly, there is no evidence that LPS is toxic to fish, and LPS from V. anguillarum acts as an immunostimulant at low concentrations (Ackerman 1995), resulting in macrophage activation and increased phagocytic activity (Solem ef al. 1995). Reasons for LPS toxicity in mammals and lack thereof in fish are unknown. It has been shown to exert effects on hsp70 induction in mammals although in these organisms it elicits a fever state which may be the reason for hsp induction. It may also have a rate limiting effect on hsp induction (Tomasovic ef al. 1991) but the relationships are still largely uncertain. Lipopolysaccharide is one of the most potent immune system alarms known and is a strong B cell mitogen. Its presence in the body results in a cascade of host immune system reactions including an increase in responsiveness of granulocytes (Behling ef al. 1979), cells that produce many highly reactive compounds such as reactive oxidative species (ROS), lysozyme, and cationic peptides. It has been suggested that ROS produced by the host as a defence against invading pathogens are equally damaging to 66 host cells and that the hsp70 response seen during the infective process may be largely due to the presence of bacterial toxins in the body (Jaquier-Sarlin ef al. 1994). In an earlier study (Ackerman et al. 2001), it was found that there was an increase in hsp70 and Cortisol during infection with V. anguillarum but it was unclear if these responses were due to presence of a toxic substance that was eliciting a stress response or to damage brought about by a live pathogen. The following study was designed to investigate this question and determine if responses seen during a live infection would be generated during a non-infectious challenge. Methods and Materials Fish and Rearing Conditions - Juvenile rainbow trout (mean weight 25.6 g ± 4.4 g) were obtained from Colebrook Trout Farm, British Columbia (B.C.), Canada and maintained at the University of British Columbia in a 1000 L tank. Fish were randomly divided into twelve 80 L tanks (2 L/min flow rate, 12°C), to a final number of 20 fish per tank (final density approximately 6.4 g/L), and were acclimated to the water system for 14 d. Fish were fed daily to satiation. Treatments - A bacterin was prepared according to Stolen ef al. (1990) from a primary isolate (Pacific Biological Station, Nanaimo, B.C., isolate number 98055) of V. anguillarum (serotype 02). Briefly, 20 h V. anguillarum cells were harvested from tryptic soy agar (TSA, supplemented with 1.5% NaCI) plates, transferred to sterile peptone-saline (P-S: 0.2 % peptone, 1.5% NaCI) and gently vortexed to make a suspension and avoid cell clumping. The suspension was then centrifuged until the supernatant was clear (1600 x g, 4°C for 25 min). The supernatant was discarded and the cell pack resuspended to a 0 .1% suspension in 0.4% formalin saline. This was held overnight at 4°C under gentle agitation. The solution was washed 3x by centrifugation in phosphate buffered saline (PBS). The cell pack was resuspended each time in roughly 10 x cell pack volume. The final suspension was estimated at 5 x 10 7 cells/mL (1 OD 5 4 onm = 10 9 cells/mL) and each anaesthetized (50 mg/L MS-222 buffered with 50 mg/L NaHC0 3 ; Syndel Laboratories, Vancouver, B.C) fish received an intraperitoneal (i.p.) injection of 100 pL of this suspension for an estimated 5 x 10 6 cells/fish. Dose was selected based on pathogen counts observed in a live infection (Ackerman and Iwama 2001). A stock solution of LPS from E. coli (Serotype 026:B6, Sigma L3755) was suspended in PBS at a concentration of 10 mg/mL. Fish received an injected dose of 35 mg/kg 67 that was previously shown to have no negative effects on salmonids (Harbell et al. 1979). Control fish received a 100 uL i.p. injection of sterile PBS. Sampling - Five fish were removed from two treatment and two control tanks every 24 h and placed in a lethal dose of anaesthetic (500 mg/L MS-222 buffered with 500 mg/L N a H C 0 3 ; Syndel Laboratories, Vancouver, B.C.). To mitigate the stress effects of netting, fish were never removed from the same tanks on consecutive days. Blood was collected from the caudal vessel with sterile, heparinized syringes and a blood smear was made for each sample for later cell enumeration. Remaining blood was centrifuged (2000 x g for 5 min) and the plasma was removed, frozen on dry ice and stored at - 70°C for later analyses of protein, Cortisol, glucose and lysozyme. Liver and head kidney tissues were quickly dissected out, frozen immediately on dry ice, and stored at - 70°C for later analysis of hsp70. Analytical Procedures - The number of leucocytes (lymphocytes, thrombocytes, monocytes, neutrophils) were calculated from the leucocyte to erythrocyte ratio obtained by examination of blood smears (Houston 1990). The total number of erythrocytes was determined from haemocytometer counts of whole blood Plasma glucose was determined using a micro-modification of the Trinder (1969) glucose oxidase method (available from Sigma (315)). Plasma protein levels were measured using the bicinchoninic acid procedure (Smith et al. 1985) and values are reported as bovine serum albumin (BSA) equivalents. Lysozyme activity in the plasma was determined by a modification of Litwack's (1955) method (Maule et al. 1996). The method was modified for use on a microplate reader and used 10 pL of plasma (or hen egg white lysozyme (HEWL) standard), and 250 pL of 0.025% w/v Micrococcus lysodeikticus suspension in 0.06 M phosphate buffer (pH 6.2). The decrease in optical density over 20 min of incubation at 25°C is reported here as pg/mL equivalent of HEW activity. Plasma Cortisol concentration for each sample was determined using an ELISA kit from Neogen (Lexington, Kentucky, USA) that had been validated for use with rainbow trout (Ackerman et al. 2001). Preparation of tissues, and dilution and determination of hsp70 was carried out by ELISA according to Ackerman et al. (2001) as a modification of Forsyth et al. (1997). Briefly, liver and head kidney samples were diluted with 10 volumes ice-cold lysis buffer (50 mM Tris, 5 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 1 pM Pepstatin A, 1 pM Leupeptin and 0.015 pM Aprotinin), sonicated on ice, vortexed, and placed back on ice. Aliquots (20 pL) of each sample were frozen immediately on dry ice and stored at - 70°C. The primary antibody (StressGen SPA-758) was used at a concentration of 1:2000. Values are reported as units relative to a positive arsenite induced control prepared according to Forsyth et al. (1997). 68 Statistical Analysis - Data were analysed using a two way ANOVA where appropriate and are presented as means ± 1 standard error (SE). Where a significant effect was noted (P<0.05) a Bonferroni's test was used to determine where differences occurred. Data from replicate tanks were not found to differ significantly and were pooled for final statistical analysis. Where data failed the normality test and transformation was unsuccessful, Dunn's method of all pairwise multiple comparison was used. The level of significance in all tests was P < 0.05. Results Plasma Protein - Plasma protein concentrations in LPS-injected fish were consistently lower compared to controls. Initially, fish treated with the bacterin (27.16 ± 0.63 mg/mL) and LPS (24.40 ± 0.37 mg/mL) both had significantly lower plasma protein concentrations compared to control fish (31.23 ± 0.88 mg/mL). While LPS treated fish had low protein levels for the duration of the study, fish receiving the bacterin had an increasing plasma protein concentration. At 4 d post treatment, bacterin treated fish (29.80 ± 0.96 mg/mL) had a similar level to controls (29.05 ± 1.49 mg/mL) and during days 5 - 7 the levels were consistently and significantly greater than controls and LPS challenged fish (Figure 3-1, Table 3-1). Only control fish showed no significant change over the duration of the challenge. Plasma Lysozyme - Plasma lysozyme activity remained relatively constant over time in control fish compared to treatment groups. Fish treated with the bacterin had a significant increase in activity between 2 and 4 d post challenge. This was followed by a reduction in activity to levels that approached those observed at day 1. Fish challenged with LPS had the most significant changes in plasma lysozyme activity with a greater activity at 24 h (26.11 ± 1.15 pg HEWL Eq) compared to controls (16.84 ± 0.85 pg HEWL Eq) and bacterin treated fish (17.38 ± 0.49 pg HEWL Eq). By 7 d post challenge there were no differences between treatment groups (Figure 3-2, Table 3-1) Plasma Cortisol - Fish exposed to LPS had the greatest changes in their plasma Cortisol levels over the course of the study. These fish had a significantly greater Cortisol concentration (140.1 ± 51.65 ng/mL) compared with both controls (18.43 ± 3.04 ng/mL) and bacterin exposed fish (6.38 ± 1.37 ng/mL) at 24 h post exposure. At day 2, LPS challenged fish (38.80 ± 4.75 ng/mL) had higher Cortisol levels than did bacterin challenged fish (8.64 ± 3.07 ng/mL) and their Cortisol levels remained elevated over bacterin and challenged fish at day 3. By 4 d, no statistical differences were apparent between groups (Figure 3-3, Table 3-1) 69 Only fish exposed to LPS had a significant change in Cortisol levels over time with a significant decrease between d1 and 62 (140.1 ± 51.65 ng/mL to 38.80 ± 4.75 ng/mL), and a further decrease between 3 and 4 d post exposure (from 28.11 ± 6.82 ng/mL to 6.68 ± 1.60 ng/mL). Plasma Glucose - Significant differences in plasma glucose concentration were seen both between and within treatment groups. Bacterin challenged fish had a consistently lower plasma glucose level than controls or LPS challenged fish, and LPS challenged fish were generally lower than controls except for at 24 h post challenge (Figure 3-4,Table 3-1). Control fish had a slight increase in glucose levels between 1 and 2 d and some variation during the remainder of the study. LPS injection resulted in a general decrease in plasma glucose between d1 and d4 with an indication of recovery to control levels by d6 and d7. Haematology -Erythrocyte numbers tended to be greater in bacterin injected fish than both control and LPS treated fish with the exception of day 5 where there was a brief increase in erythrocyte numbers in the LPS group, although only significantly so compared to controls. Only LPS resulted in a significant change in erythrocyte numbers over time (Table 3-3). All three treatments resulted in a general increase in total leucocytes over time but the trend was minimized for fish receiving the bacterin. However, LPS led to significantly fewer leucocytes than bacterin injected fish between days 1 and 4. The differential leucocyte counts showed few differences between treatments with respect to neutrophils (Table 3-3), monocytes or thrombocytes (Table 3-4). At day 1 there was a greater circulating concentration of neutrophils in fish receiving the bacterin and at day 4 there was a greater concentration of thrombocytes in bacterin injected versus LPS injected fish, neither of which differed significantly from controls. There were no differences in circulating levels of monocytes at any time point. Numbers of circulating lymphocytes tended to be lower in the LPS group than sham injected or bacterin injected fish between 1 and 4 d (Table 3-3). Hsp70 - In response to LPS, head kidney (Figure 3-6) tissue had a gradual increase in hsp70 levels over the first few days of challenge with levels decreasing towards the end of the challenge. However, the only significant change over time was between 3 and 7 d. Bacterin injected fish tended to have lower hsp70 levels in the head kidney but this was not statistically significant compared to controls at any time point. Only LPS treated fish had a significant change in levels over time (Table 3-2). There was a marked decrease in liver hsp70 levels in fish injected with either LPS or the bacterin at 2 and 3 d post challenge (Figure 3-7). Fish injected with bacterin also showed a significant decrease in hsp70 levels at day 6. While sham injected fish had no change in hsp70 levels over time, both treatment groups did (Table 3-2). 70 Q saline A. E. coli LPS (35 mg/kg) • bacterin (106 cells/fish) 0 —r-2 - r -3 — r -4 5 i 6 —r -7 Time (d) Figure 3-1. Mean (± SE) plasma protein concentration over 7 days following injections with a formalin killed bacterin, 35 mg/kg LPS, or saline. Statistical differences between groups within time periods are denoted by superscripts (P < 0.05). 71 Figure 3-2. Mean (± SE) plasma lysozyme activity (B) over 7 days following injections with a formalin killed bacterin, 35 mg/kg LPS, or saline. Statistical differences between groups within time periods are denoted by superscripts (P < 0.05). 72 Figure 3-3. Mean (± SE) plasma Cortisol concentration over 7 days following injections with a formalin killed bacterin, 35 mg/kg LPS, or saline. Statistical differences between groups within time periods are denoted by superscripts (P < 0.05). 73 Figure 3-4. Mean (± SE) plasma glucose concentration over 7 days following injections with a formalin killed bacterin, 35 mg/kg LPS, or saline. Statistical differences between groups within time periods are denoted by superscripts (P < 0.05). 74 3e+5 n 3e+5 O saline A E. coli LPS (35 mg/kg) • bacterin (106 cells/fish) 2e+5 co CD I T 2e + 5 o LU 1e+5 5e+4 2 —r -3 ~i 5 i 6 Time (d) Figure 3-5. Mean (± SE) erythrocyte numbers over 7 days following injections with a formalin killed bacterin, 35 mg/kg LPS, or saline. Statistical differences between groups within time periods are denoted by superscripts (P < 0.05). 75 60 - i O saline ^ 50 - A E. coli LPS (35 mg/kg) ' c • bacterin (106cells/fish) CD X 10 T 1 1 1 1 1 r 1 2 3 4 5 6 7 T ime (d) Figure 3-6. Mean (± SE) hsp70 levels in head kidney tissue over 7 days following injections with a formalin killed bacterin, 35 mg/kg LPS, or saline. Statistical differences between groups within time periods are denoted by superscripts (P < 0.05). 76 O saline A E. coli LPS (35 mg/kg) 1 2 3 4 5 6 7 Time (d) Figure 3-7. Mean (± SE) hsp70 levels in liver tissue over 7 days following injections with a formalin killed bacterin, 35 mg/kg LPS, or saline. Statistical differences between groups within time periods are denoted by superscripts (P < 0.05). 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The responses generated by the presence of a killed bacterin based on an identical strain of V. anguillarum used for live challenges (Ackerman et al. 2001) and LPS from E coli indicate that there are measurable stress responses to non-lethal immune challenges. Because of a high degree of homology, LPS from E. coli has been successfully used in studies on trout to examine immunological function (Bowers et al. 2000; Brubacher ef al. 2000). There was a consistent difference in haematological effects between LPS treatment and bacterin-treated fish or controls. In general, with the exception of thrombocyte and monocyte numbers, bacterial LPS exerted a significantly greater overall effect on blood cell numbers than did saline or bacterin injections. There was a greater proportion of erythrocytes/mL blood in fish challenged with the bacterin at all time points except day 5. Such high erythrocyte levels are often interpreted as due to haemoconcentration/dehydration and attributed to stress. LPS treated fish had a brief but significant increase in erythrocyte levels at day 5, but variation was large and this difference is likely an artefact. The majority of vaccine and infection studies show very few differences with respect to differential counts of blood cell numbers. In the present study, when cell numbers were converted into proportions based on haemocytometer counts, some differences became apparent. There were few differences between total leucocyte counts although LPS challenged fish tended to have lower numbers over the first few days following challenge. Such variable responses as were seen in leucocyte counts of LPS treated fish can be interpreted as an initial stress mediated decrease in leucocytes. Such decreases in response to stressful conditions have been reported (McLeay 1975). Viruses and bacteria have been recorded at up to 10 9/mL of seawater (Hennes et al. 1995). The vast majority of aquatic bacterial pathogens are Gram-negative and LPS is a Gram-negative bacterial endotoxin generally accepted to be a powerful pyrogen and mitogen as well as a strong polyclonal B-cell stimulator (Anderson 1992) that has profound effects on haematopoietic tissues in mammals (Behling ef al. 1979). The lipid-A region is the toxic portion and is the least variable. Studies using LPS to investigate its effects on immunomodulation in fish have demonstrated that it is capable of enhancing macrophage phagocytic activity, migratory activity, and superoxide anion production both in vivo and in vitro (MacArthur ef al. 1985; Salati ef al. 1987; Solem ef al. 1995). Fish are predisposed to react to 82 LPS because the lipid-A fraction is structurally similar in all Gram-negative species and it is against the lipid-A that immunomodulatory activity is thought to occur (Jacobs 1991). The endotoxin is likely recognized quickly because of a long evolutionary process whereby animals have developed mechanisms to detect such common and highly conserved chemical moieties. This predisposition means the immune system interprets its introduction as a bacterial invasion as LPS is the most potent immune alarm signal known. However, neither the nature of LPS stimulation nor the B-cell receptor is known. In mammals LPS is extremely toxic; high concentrations cause tissue injury, intravascular injury and shock, but it does not induce similar toxic effects in fish (Harbell et al. 1979) where doses of up to 355 mg/kg body weight fail to produce clinical signs including haemodynamic changes even though most was absorbed by the blood. It could be that fish have an extremely efficient system in place to detoxify LPS because they have evolved in an environment where they are regularly challenged by Gram-negative pathogens, or it could be that cellular responses to LPS are simply not as developed as in mammals. More likely it is a combination of these. Cortisol and glucose are often used in combination as an indication of a generalized stress response and while there were significant differences between treatment groups during the first three days post challenge, the Cortisol levels seen in LPS treated fish at day 1 were the only levels that would be generally considered to be elevated outside of the normal range. While there were significant differences between bacterin and sham injected fish, both of these groups had quite low levels of Cortisol in circulation at all points in time. The plasma glucose data support the Cortisol data suggesting that there was likely a short term stress response to the presence of LPS in circulation. Fish immunized with the bacterin had plasma glucose levels that were significantly lower than in controls at all but day 7 and these levels were also significantly lower than jn fish receiving LPS injection. Basal levels of glucose in trout are usually in the range of 60-80 g/dL (Himmick et al. 1990) and stress responses result in a transient increase as energy resources are redistributed to address the energetic requirements of responding to a threat. The drop in glucose level in the two treated groups may indicate a reduction in their ability to cope with the stressor as the lower level could indicate a reduced ability to mobilize energy. It is interesting to note that the bacterin immunized fish also had a consistently lower total plasma protein concentration than the other two groups of fish while at the same time having an overall greater lysozyme activity suggesting a shift in composition of plasma proteins. That the bacterin produced such different results in the head kidney compared to the LPS was unexpected as the antigen processing would still be expected to produce some 83 degree of oxidative reactants. Perhaps the formalin killed bacterin was less reactive than live pathogen due to formalin treatment, but this is not known. While the bacterin would have LPS associated with the cells, it would not contain a level as great as that used in LPS immunized fish and it is possible that the high LPS concentration provided a greater stimulation to the immune system than did killed V. anguillarum cells. When applied intravenously to fish, LPS is rapidly cleared from blood, primarily localizing in the kidney (anterior and posterior) and to a lesser degree in liver, heart and spleen tissues (Stensvag et al. 1999). Endotoxin activates the complement system (Boesen etal. 1999) and elicits production of TNF-a, interleukins (IL-1, IL-6), prostaglandins (PGE2), free radicals (02-), and hydrogen peroxide (Rietschel et al. 1992). This response occurs in salmonids irrespective of whether LPS is from a Vibrio species familiar to fish or from an alien bacterial species (reviewed in Raa 1996). Many compounds produced by the immune system in response to a pathogenic challenge are damaging not only to cells at which they are directed, but also to self, with damage occurring through oxidative processes. There were treatment effects with respect to hsp70 in both liver and head kidney tissues but changes were not consistent. In the head kidney, LPS treated fish had a significantly greater hsp70 level than bacterin injected fish from 1 to 3 d but differed from controls only at 3 d. While slightly lower, the bacterin immunized fish were not significantly different from controls. In a live challenge levels of hsp70 increased steadily between 1 and 6 d and were significantly greater than controls at 4 through 7 d (Ackerman et al. 2001), a time that roughly corresponded to an increase in concentration of bacteria in the system, and therefore likely an increased level of bacterial LPS in circulation. The changes in hsp70 concentration in liver are difficult to reconcile considering changes that occurred during a live challenge. Both LPS and bacterin treatments resulted in a significant decrease in hsp70 levels in liver between 1 and 2 d. Levels in both groups remained low between 2 and 3 d and returned to control levels by day 3. Bacterin challenged fish had another decrease between d5 and d6 and then again returned to control levels on day 7. In a live challenge, hsp70 had a steady increase but was not significantly different from controls except at 5 and 6 d, returning to control levels by 7 d. Clinical infection is characterized by erythema, haemorrhaging and anaemia therefore this difference may be related to physical damage arising from live infection and resulting in an increased hsp production in an attempt to repair damage, but this cannot be confirmed with the current data. Decreased tissue levels of the protein are not necessarily an indication of a reduced synthesis but may represent an increased turnover rate such that breakdown outstrips synthesis. Again, without a 84 measurement of the protein breakdown in the current study, this cannot be conclusively addressed. Vaccines have been used for decades to combat infectious agents with varying degrees of success. Most studies obviously focus on protective effects of traditional and novel vaccines, and while there have been some studies on effects of vaccination on growth of fish (Lillehaug et al. 1992; Midtlying et al. 1998), few studies have examined the physiological responses to bacterial components or vaccines. Studies that have examined the responses of juvenile rainbow trout to vaccination both with and without adjuvants (Ackerman 1995; Ackerman et al. 2000) found that LPS exerted significant effects on many aspects of the fish's physiology from an increased metabolic rate to increases in the somatic index of head kidney tissue. LPS was also found to result in a drop in leucocrit, an increase in haematocrit, and an increase in lysozyme activity. However, the LPS in that study was microencapsulated with a bacterin and therefore released over a period of time. In the present study, LPS was injected as a bolus which likely gave rise to some of the early responses, namely increased Cortisol concentration and lysozyme activity. Among the most successful vaccines for salmonid fishes are vibrio bacterins administered by intraperitoneal injection, oral administration, or bath immersion. Application of such bacterins to invertebrates has also been shown to elicit protection against subsequent infections (Itami ef al. 1989; Home ef al. 1995). Since invertebrates do not have a specific immune response, it is still largely unknown what components of V. anguillarum cells stimulate the non-specific responses, but there is a reasonable probability that LPS may be the immunostimulant responsible for the protective effects of such vaccines (Velji ef al. 1992; Steine ef al. 2001). This study was designed in an attempt to discern whether the responses seen to a live pathogenic challenge with V. anguillarum could be wholly or partially attributable to the simple presence of the pathogen in a killed form, or to a known mitogenic component of Gram-negative bacteria, LPS. It is apparent from the results that the responses to a live disease challenge cannot be solely attributed to the presence of an active infection. Immunizations and the incorporation of immunostimulants (such as LPS in fish) are primarily designed to enhance immune responses and protect animals from disease. Vaccine developers are constantly attempting to find the best means to create functional vaccines but it is obvious that more studies must be undertaken to examine other influences that therapeutants as well as immunomodulators such as LPS have on the delicate homeostatic balance. 85 References Ackerman, P. A. 1995. Effects of adjuvanted Aeromonas salmonicida vaccines on growth, oxygen consumption, and selected haematological variables in juvenile rainbow trout (Oncorhynchus mykiss). Department of Animal Science. Vancouver, BC, Canada, University of British Columbia: 94 p. Ackerman, P. A. and G. K. Iwama. 2001. Physiological and cellular stress responses of juvenile rainbow trout to vibriosis infection. Journal of Aquatic Animal Health 13: 173-180. Ackerman, P. A., J. C. Thornton and G. K. Iwama. 2000. Physiological and immunological effects of adjuvanted Aeromonas salmonicida vaccines on juvenile rainbow trout. Journal of Aquatic Animal Health 12:157-164. Anderson, D. P. 1992. Immunostimulants, adjuvants, and vaccine carriers in fish: applications to aquaculture. Annual Review of Fish Diseases 2: 281-307. Behling, U. H. and A. Nowotny. 1979. Immunostimulation by LPS and its Derivatives. Immunomodulation by Bacteria and Their Products. H. Friedman, T. W. Klein and A. Szentivanyi. New York, Plenum Press: 165-179. Boesen, H. T., K. Pedersen, J. L. Larsen, C. Koch and A. E. Ellis. 1999. Vibrio anguillarum resistance to rainbow trout (Oncorhynchus mykiss) serum: role of O-antigen structure of lipopolysaccharide. Infection and Immunity 67: 294-301. Bowers, J. M., A. Mustafa, D. J. Speare, G. A. Conboy, M. Brimacombe, D. E. Sims and J. F. Burka. 2000. The physiological response of Atlantic salmon, Salmo salar L., to a single experimental challenge with sea lice, Lepoptheirus salmonis. Journal of Fish Diseases 23: 165-172. Brubacher, J. L., C. J. Secombes, J. Zou and N. C. Bols. 2000. Constitutive and LPS-induced gene expression in a macrophage-like cell line from the rainbow trout (Oncorhynchus mykiss). Developmental and Comparative Immunology 24: 565-574. Forsyth, R. B., E. P. M. Candido, S. L. Babich and G. K. Iwama. 1997. Stress protein expression in coho salmon with Bacterial Kidney Disease. Journal of Aquatic Animal Health 9: 18-25. Harbell, S. C , H. O. Hodgins and M. H. Schiewe. 1979. Studies on the pathogenesis of vibriosis in coho salmon Oncorhynchus kisutch (Walbaum). Journal of Fish Diseases 2: 391-404. Hennes, K. P. and C. A. Suttle. 1995. Direct counts of viruses in natural waters and laboratory cultures by epifluorescence microscopy. Limnology and Oceanography 40: 1050-1055. Himmick, B. A. and J. G. Eales. 1990. Acute correlated changes in plasma T4 and glucose in physically disturbed cannulated rainbow trout, Oncorhynchus mykiss. Comparative Biochemistry and Physiology 97A: 165-167. Home, M. T., M. Poy and P. Pranthanpipat. 1995. Control of vibriosis in black tiger shrimp, Penaus monodon by vaccination. The Third Asian Fisheries Forum. Asian Fisheries Society. Manila, Philippines. Houston, A. H. 1990. Blood and circulation. Methods for fish biology. C. B. Schreck and P. B. Moyle. Bethesda, Maryland, American Fisheries Society: 273-334. 86 Itami, T., M. Takahashi and T. Nakamura. 1989. Efficacy of vaccination against vibriosis in cultured kuruma prawns Peneaus japonicus. Journal of Aquatic Animal Health 1: 238-242. Jacobs, D. M. 1991. Immunomodulatory effects of bacterial LPS. Journal of Immunopharmacology 3: 119-132. Jaquier-Sarlin, M. R., K. Fuller, A. T. Dinh-Xuan, M. J. Richard and B. S. Polla. 1994. Protective effects of hsp70 in inflammation. Experientia 50: 1031-1038. Lillehaug, A., T. Lunder and T. T. Poppe. 1992. Field testing of adjuvanted furunculosis vaccines in Atlantic salmon, Salmo salar L. Journal of Fish Diseases 15: 485-496. Litwack, G. 1955. Photometric determination of lysozyme activity. Proceedings for the Society for Experimental Biology and Medicine 89: 401-403. MacArthur, J. I., A. W. Thomson and T. C. Fletcher. 1985. Aspects of leucocyte migration in the plaice Pleuronectes platessa L. Journal of Fish Biology 27: 667-676. Maule, A. G., R. Schrock, C. Slater, M. S. Fitzpatrick and C. B. Schreck. 1996. Immune and endocrine responses of adult chinook salmon during freshwater immigration and sexual maturation. Fish and Shellfish Immunology 6: 221-233. McLeay, D. J. 1975. Sensitivity of blood cell counts in juvenile coho salmon (Oncorhynchus kisutch) to stressors including sublethal concentrations of pulp mill effluent and zinc. Journal of the Fisheries Research Board of Canada 32: 2257-2364. Midtlying, P. J. and A. Lillehaugh. 1998. Growth of Atlantic salmon Salmo salar after intraperitoneal administration of vaccines containing adjuvants. Diseases of Aquatic Organisms 32: 91-97. Raa, J. 1996. The use of immunostimulatory substances in fish and shellfish farming. Reviews in Fisheries Science 4: 229-288. Rietschel, E. T. and H. Brade. 1992. Bacterial endotoxins. Scientific American (August): 26-33. Salati, F., M. Hamaguchi and R. Kusuda. 1987. Immune response of red sea bream to Edwardsiella tarda antigens. Fish Pathology 22: 93-98. Smith, P. K., R. I. Krohn, G. T. Hermanson, A. K. Mallia, F. H. Gartenr, M. D. Provenzano, E. K. Fujimoto, N. M. Goeke, B. J. Olson and D. C. Klenk. 1985. Measurement of protein using bicinchoninic acid. Analytical Biochemistry 150: 76-85. Solem, S. T., J. B. Jorgensen and B. Robertsen. 1995. Stimulation of respiratory burst and phagocytic activity in Atlantic salmon (Salmo salar, L.) macrophages by lipopolysaccharide. Fish and Shellfish Immunology 5: 475-491. Steine, N. O., G. O. Melingen and H. I. Wergeland. 2001. Antibodies against Vibrio salmonicida lipopolysaccharide (LPS) and whole bacteria in sera from Atlantic salmon (Salmo salar L.) vaccinated during the smolting and early post-smolt period. Fish and Shellfish Immunology 11: 39-52. Stensvag, K., J. Bogwald, B. Smedsrod and T. O. Jorgensen. 1999. Distribution of intravenously injected A-layer protein and lipopolysaccharide (LPS) from Aeromonas salmonicida in Atlantic salmon, Salmo salar L. Fish and Shellfish Immunology 9: 591-607. 87 Stolen, J. S., T. C. Fletcher, D. P. Anderson, B. S. Roberson and W. B. van Muiswinkel, Eds. 1990. Techniques in Fish Immunology. Fish Immunology Technical Communications. Fair Haven, NJ, SOS Publications. Tomasovic, S. P. and J. Klostergaard. 1991. Bacterial-endotoxin lipopolysaccharide modulates synthesis of the 70 kDa heat-stress protein family. International Journal of Hyperthermia 7: 643-651. Trinder, P. 1969. Determination of glucose in blood using glucose oxidase with an alternative oxygen acceptor. Annals of Clinical Biochemistry 6: 24-27. Velji, M. I., L. J. Albright and T. P. T. Evelyn. 1992. Immunogenicity of various Vibrio ordalii lipopolysaccharide fractions in coho salmon Oncorhynchus kisutch. Diseases of Aquatic Organisms 12: 97-101. 88 Section ill. The effect of Cortisol increase during infection The study in Chapter 3 provided new insights into stress responses in relation to non-lethal immunological challenge such as application of vaccines or immunomodulators. Injection with LPS was observed to result in an increase in hsp70 in head kidney tissue while liver levels decreased significantly following exposure to either LPS or a killed pathogen. Cortisol increased in response to acute infection in a live challenge study (Chapter 2) as well as during the early stages of a non-lethal immune challenge (Chapter 3). Other researchers have demonstrated a decrease in hsp70 response during physiological stress and it was unknown if the significant increase in Cortisol levels in the live challenge had any effect on hsp70 levels during an infection. Although there was an increase in hsp70 during infection, did this represent a moderation of the response? In order to determine if Cortisol is a required component of response to acute bacterial infection, a study was undertaken to attempt to remove physiological effects of Cortisol during an infection by using a compound known to block the Cortisol response, RU-486. 89 CHAPTER 4 Use of the Cortisol blocker RU-486 in fish health studies. Introduction All organisms are regularly faced with challenges that threaten homeostasis. Although neuro-endocrine responses to stress have been extensively studied in fish, information about the cellular stress response is not as readily available and relationships between the two are still being elucidated warranting further studies to clarify the complex interactions between hormones and stressors on hsp expression in fish. Glucocorticoids are agents that promote metabolic breakdown of carbohydrates, proteins and lipids to mobilize energy reserves when the body is placed under duress. Cortisol is the most widely studied of these stress related hormones and is responsible for a host of biological events such as effects on bone, cell growth, hydromineral balance, behaviour and feeding (see Wendelaar Bonga 1997). Cortisol enters target cells through the membrane and binds to cytoplasmic receptors following which it is translocated to the nucleus where it initiates specific changes in DNA transcription and protein synthesis (Mommsen et al. 1999). Physiological effects of Cortisol are mediated by the glucocorticoid receptor (Wendelaar Bonga 1997) which, in turn, is dependent on hsps for its assembly, transport, and function (Pratt 1993). One of the most biologically significant events any organism faces on a regular basis is immunological challenge. The immune system is constantly functioning to recognize and rid the body of any foreign materials and events that unfold during an immune response are violent in nature at the cellular level. Chronic Cortisol elevation is known to have negative influences on disease resistance (Maule et al. 1989), and interfere with lymphocyte function (Tripp et al. 1987). Glucocorticoids have a variety of inhibitory effects on immunity due to actions on cells of the immune system and Cortisol has been shown to interfere with lymphocytes by inhibiting their transformation into antibody producing cells and by inhibiting release or synthesis of cytokines required to stimulate antibody production (reviewed by Colombo et al. 1989; Marx 1995). In a previous study, we (Ackerman et al. 2001) showed an increase in plasma Cortisol concentration over time that followed the dynamics of an acute infection but it is not known if this is a contributing factor to deterioration of those fish succumbing to disease or if the subsequent decline reflects fish in a state of recovery. In the few studies which have examined the infective process in fish from a physiological perspective, 90 changes have generally been observed only at the extreme end of an infection (Laidley et al. 1988). Several studies have examined relationships between Cortisol and physiological responses through use of both Cortisol augmentation (Pottinger 1990; Specker ef al. 1994; Vijayan ef al. 1997) and blockage (Vijayan ef al. 1994; Reddy ef al. 1995). To date however, no studies have examined the effects of the Cortisol blocker RU-486 on the physiological responses to an infection in fish. RU-486 is a steroid analogue and a powerful Cortisol antagonist that has been shown to displace Cortisol from the glucocorticoid receptor (Pottinger 1990). By blocking the Cortisol receptor, RU-486 abolishes the cascade of events brought about by an increase in Cortisol. The compound has been used with relative frequence in physiological studies designed to examine functions of Cortisol and it has been demonstrated that when applied in slow release implants, it does not prevent stress related release of Cortisol. Further, it has been reported to not have any effect on plasma Cortisol concentrations (Reddy ef al. 1995). This study was designed to examine the relationship between the neuro-endocrine and cellular stress responses during an acute bacterial infection. Rainbow trout (Oncorhynchus mykiss) were implanted by injection with the exogenous Cortisol blocker RU-486 and subsequently challenged with the pathogen Vibrio anguillarum to examine the relationship between Cortisol concentration and physiological responses to immunological challenge Methods and Materials Fish and Rearing Conditions - Juvenile rainbow trout (mean weight 21.2 g ± 3.6 g) were obtained from Colebrook Trout Farm, British Columbia (B.C.), Canada and maintained at the University of British Columbia in two 1000 L tanks. Fish were randomly divided into twelve 80 L tanks (2 L/min flow rate, 11°C), to a final number of 70 fish per tank (final density approximately 18.4 g/L), and were acclimated to the water system for 14 d. Fish were fed daily to satiation with a commercial salmonid feed (Moore Clarke, BC). Implantation - Fish were lightly anaesthetized (50 mg/L MS-222, 50 mg/L NaHC0 3 ) and given a single intraperitoneal injection of either warm hydrogenated coconut oil/vegetable oil alone (1:1) or coconut oil/vegetable oil containing RU-486 (100 mg/kg fish weight). When the oil mixture was injected it formed a semi-solid pellet in the intraperitoneal cavity. The protocol was carried out according to Vijayan ef al. (1994). Fish received an injection of approximately 100 pL of the carrier or carrier plus RU-486 based on fish weight. Following 91 implantation, fish were returned to their respective tanks for 24 h recovery prior to disease challenge. Disease Challenge - A primary isolate (Pacific Biological Station, Nanaimo, B.C., isolate number 98055) of V. anguillarum (serotype 02) was obtained from wild chum salmon (O. keta) that had died from natural exposure to the pathogen. Twenty hour V. anguillarum cells were harvested from tryptic soy agar (TSA, supplemented with 1.5% NaCl) plates, transferred to sterile peptone-saline (P-S: 0.2 % peptone, 1.5% NaCl) and vortexed to make a suspension. The concentration of the V. anguillarum suspension was estimated from absorbance measurements made at 540 nm (1 O D 5 4 0 estimated to contain 109 cells/mL). The suspension was diluted to an estimated concentration of 1 x 10 e cfu/mL (actual dose 1.04 x 10 5 cfu/mL determined by drop plate counts). Fish from three tanks of each of the two challenge groups were injected with 100 uL of the bacterial suspension (10 4cfu/fish); fish from the sham control and RU-486 control tanks received the peptone-saline carrier only (100 pL/fish). Two tanks of each group were used for daily sampling of fish and the remaining tank was used for mortality data. Five fish were removed from two of each of the sampling tanks every 24 h and placed in a lethal dose of anaesthetic (500 mg/L MS-222 buffered with 500 mg/L NaHC0 3 ; Syndel Laboratories, Vancouver, B.C.). Blood was collected from the caudal vessel with sterile, heparinized syringes for determination of viable pathogen counts, haematocrit, and haemoglobin. The remaining blood was centrifuged (2000 x g for 5 min) and the plasma was removed, frozen on dry ice and stored at -70°C for later analyses of protein, Cortisol and glucose concentration, and lysozyme activity. Liver and head kidney tissues were quickly dissected out, frozen immediately on dry ice, and stored at -70°C for later analysis of hsp70. Analytical Procedures - Blood haemoglobin levels were measured as cyanmethaemoglobin with modified Drabkin's reagent and a standard containing human haemoglobin (Sigma Chemical Co. kit 525-A). Haematocrit (% red blood cells) was determined using the method described by Houston (1990). Viable pathogen numbers were determined according to the method described by Balfry et al. (1997). Plasma glucose levels were determined using a microtized modification of the Trinder (1969) glucose oxidase method (Sigma Chemical Co. kit 315) to use smaller sample volumes, while plasma protein levels were determined using the bicinchoninic acid procedure (Smith et al. 1985) and values are reported as bovine serum albumin (BSA) equivalents. Plasma Cortisol concentrations were determined using a commercially available enzyme-linked immunosorbent assay (ELISA) kit (Neogen Corp. Lexington, KY). Lysozyme activity in plasma was determined by a modification of Litwack's (1955) method (Maule et al. 1996). The method was modified for use on a microplate 92 reader and used 15 pL of plasma (or hen egg white lysozyme (HEWL) standard), and 250 pL of 0.025% w/v Micrococcus lysodeikticus suspension in 0.06 M phosphate buffer (pH 6.2). The decrease in optical density over 20 min of incubation at 25°C is reported here as pg/mL equivalent of HEWL activity. Leucocytes were categorized from blood smears as lymphocytes, neutrophils, monocytes and thrombocytes based on morphological characteristics described by Yasutake and Wales (1983) and Houston (1990). Liver and head kidney samples were diluted with 10 volumes ice-cold lysis buffer (50 mM Tris, 5 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 1 pM Pepstatin A, 1 pM Leupeptin and 0.015 pM Aprotinin), sonicated on ice, vortexed, and placed back on ice. Aliquots of each sample were frozen immediately on dry ice and stored at - 70°C. Preparation of tissues, and dilution and determination of hsp70 was carried out by ELISA according to Forsyth et al. (1997) and reported as units relative to a positive arsenite induced control. The hsp70 antibody used was prepared as outlined in Forsyth et al. (1997). Four mortality control tanks were maintained and monitored daily for three weeks following the challenge. Fish that died were assayed for the presence of V. anguillarum by streaking aseptically removed kidney swabs onto TSA supplemented with 1.5% NaCI. Creamy white bacterial colonies that Gram-stained negative and displayed motility under the microscope were identified as V. anguillarum. Statistical Analysis - Data were analysed using a two way ANOVA where appropriate and are presented as means ± 1 standard error (SE). Where a significant effect was noted (P<0.05) a Bonferroni's test was used to determine where differences occurred. Data from replicate tanks were not found to differ significantly and were pooled for final statistical analysis. Where data failed the normality test and transformation was unsuccessful, Dunn's method of all pairwise multiple comparison was used. The level of significance in all tests was P < 0.05. Results Mortality - There was no statistical difference (P=0.114) between disease challenged fish receiving RU-486 injection (76% mortality) and those fish that received the pathogen challenge and carrier alone (61% mortality) (Figure 4-1). Plasma Cortisol - Circulating Cortisol levels were greater in both groups that received the Cortisol blocker than in fish that received the sham carrier. Fish receiving sham carrier + V. anguillarum had a gradual increase in circulating Cortisol levels with a significantly 93 greater concentration than sham controls at day 4 with a subsequent decrease at day 5. Sham control fish had a consistently low level of Cortisol while RU-486 + V. anguillarum challenged fish had a significantly greater Cortisol concentration at the final sampling period compared to all other treatment groups (Figure 4-2). RU-486 controls had consistently higher plasma Cortisol concentrations than did sham controls throughout most sampling periods. Neither the sham control nor RU-486 control fish had any significant change in Cortisol levels over the sampling period (Table 4-1) but both disease challenged groups did. RU-486 challenged fish had a transiently decreased Cortisol level at day 2 and subsequently had a greater concentration than any other group. Sham challenged fish had a gradual but significant increase in Cortisol levels between d1 and d4 with a subsequent decrease to initial levels by day 5. Plasma Glucose - There were no significant differences in plasma glucose among treatment groups at any sampling period (Table 4-1) and only RU-486 challenged fish had a change in plasma glucose levels over the duration of the sampling period. Plasma Protein - Fish injected with both RU-486 and V. anguillarum had a gradual decrease in plasma protein levels between 1 and 4 d. These levels were significantly below sham controls between 3 and 4 d, and below all other groups at 4 d post challenge. At day 4 both treatment groups and RU-486 controls had significantly lower concentrations than sham controls. RU-486 controls had a consistently lower protein level than sham control fish (Figure 4-3). Within treatment groups, neither sham control nor RU-486 control fish displayed any differences over the duration of the study (Table 4-1). Both challenged groups had a decrease in plasma protein between 1 and 4 d followed by a return to initial levels by day 5. Plasma Lysozyme Activity - Plasma lysozyme levels were inconsistent through the study and showed no clear pattern. Some differences were seen at 1, 5 and 7 d; at 1 and 5 d post challenge, levels were significantly higher in RU-486 challenged fish (Table 4-1) while sham challenged fish had a significantly lower activity level than other groups at day 7. All groups had significant differences in activity over the duration of the sampling period. Haematology - Haemoglobin levels in RU-486 challenged fish were consistently lower than sham controls for the first 4 d post challenge but returned to control levels by day 5. These differences were only significant at 1 and 3 d (Figure 4-3). Only sham challenged fish had a significant change in haemoglobin concentration over the sampling period with an initial decrease between 1 and 3 d followed by a significant increase to 5 - 7 d (Table 4-2). Haematocrit concentrations fluctuated throughout the sampling period in all treatments (Table 4-2) but RU-486 challenged fish had a gradual decline between 1 and 4 d returning to control 94 levels at day 5 (Figure 4-5). Erythrocyte numbers were consistent in sham controls and, although slightly lower, RU-486 controls over the duration of the sampling period (Table 4-2). RU-486 controls had significantly fewer circulating erythrocytes than sham controls at 3 and 5 d (Figure 4-6). Sham challenged fish initially had significantly greater circulating erythrocytes than all other groups but this then declined to significantly fewer than all treatment groups by day 4. RU-486 challenged fish had a transient decrease in cell numbers to lower than sham controls between 3 and 5 d. At day 7 both challenged groups had significantly fewer circulating erythrocytes than both control groups. Few differences were observed in leucocyte population composition or numbers and there was no clear pattern to the differences. Only RU-486 controls had a change in total leucocyte numbers over the sampling period, with a significant decrease to day 5 and a subsequent increase by day 7 (Table 4-2). There was a similar trend in the two challenged groups but the differences between sampling points were not significant. At day 5 both RU-486 controls and RU-486 challenged fish had significantly fewer leucocytes than controls but were not significantly different from each other. Individual leucocyte populations showed no differences between sampling times and few differences between groups within sampling periods were observed. At 4 and 5 d both RU-486 controls and RU-486 challenged fish had significantly fewer lymphocytes than controls but were not significantly different from each other. This pattern was also observed in the neutrophil population at day 6. Hsp70 - All treatment groups had a decreasing trend in liver hsp70 levels between 1 and 2 d. However, sham control hsp70 levels remained relatively constant with no significant change throughout the sampling period (Table 4-4) and the change in hsp70 between 1 and 2 d was significant in only RU-486 challenged fish. RU-486 challenged fish had significantly lower liver hsp70 levels at 2 and 3 d post challenge compared to all other treatments (Figure 4-7) and these were below both control groups at day 4. There was a transient decrease in hsp70 levels in RU-486 controls at day 5 compared to controls and sham challenged fish. At the final sampling time both RU-486 controls and RU-486 challenged fish had higher levels of hsp70 than initial values. Head kidney hsp70 levels in RU-486 challenged fish were significantly below both control groups at day 5 (Figure 4-8) and this was the only point at which groups differed within a sampling time. However, there was an observed change in levels within all groups including the controls across sampling times (Table 4-4). 95 O oil + saline • RU486 + saline O Oil + V. ang 0 RU-486 + V. ang - n - f T - ^ - n - n - n - n - r ^ ^ 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Time (d) Figure 4-1 . Percent cumulative mortality in juvenile rainbow trout challenged with 10 4 cfu/fish V. anguillarum in the presence and absence of the Cortisol blocker RU-486. 96 500 -i O oil + saline • RU486 + saline <0> oil + V. ang 0 RU486 + V. ang Time (d) Figure 4-2. Plasma Cortisol concentration in fish challenged with and without RU-486 and V. anguillarum. Means are presented ± SE. Letters denote statistical differences within each sampling period (P < 0.05) 9 7 O oil + saline • RU486 + saline <^ > oil + L. ang 0 RU486 + L ang 6 —r-2 —I -3 4 Time (d) Figure 4-3. Plasma protein concentration in fish challenged with and without RU-486 and V. anguillarum. Means are presented ± SE. Letters denote statistical differences within each sampling period (P < 0.05) 98 Q oil + saline • RU486 + saline < ^ oil + V. ang Q RU486 + V. ang ~T~ 2 ~~r~ 4 5 6 ~T~ 7 Time (d) Figure 4-4. Haemoglobin concentrations in fish challenged with and without RU-486 and V. anguillarum. Means are presented ± SE. Letters denote statistical differences within each sampling period (P < 0.05) 99 5 0 - i O oil + saline • RU486 + saline O oil + V. ang 0 RU486 + V. ang 40 -O CO. 0 J 1 1 1 i 1 1 r 1 2 3 4 5 6 7 Time (d) Figure 4-5. Haematocrit concentrations in fish challenged with and without RU-486 and V. anguillarum. Means are presented ± SE. Letters denote statistical differences within each sampling period (P < 0.05) 100 3e+5 2e+5 A O oil + saline • RU486 + saline <C> oil + V. ang 0 RU486 + V. ang 2e+5 1e+5 5e+4 3 4 5 Time (d) Figure 4-6. Erythrocyte concentrations in fish challenged with and without RU-486 and V. anguillarum. Means are presented ± SE. Letters denote statistical differences within each sampling period (P < 0.05) 101 O oil + saline • RU486 + saline <£> oil + V. ang 0 RU486 + V. ang 3 4 Time (d) 7 Figure 4-7. Liver hsp70 tissue levels in fish challenged with and without RU-486 and V. anguillarum. Means are presented ± SE. Letters denote statistical differences within each sampling period (P < 0.05) 102 O oil + saline • RU486 + saline O oil + V. ang 0 RU486 + V. ang - T -2 i 4 Time (d) -r~ 5 Figure 4-8. Head kidney hsp70 tissue levels in fish challenged with and without RU-486 and V. anguillarum. Means are presented ± SE. 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XJ <o ro - c 0 c Zi 0 > to 0 CM O X co X l o CM o CO CM X CD CO •sr CM O ai CNI LO X LO CO X I s -o I s-^r CNI o D) C co X •sr LO CO c co CO 00 •sr 1 Z5 Cd CD c co 0 JO ro > 107 Discussion The current study was designed to investigate the role of Cortisol in the infective process by removing the hormone's effects through use of RU-486. It has been demonstrated that Cortisol has a dampening effect on the hsp70 response to stress (Ackerman et al. 2000; Basu et al. 2001; Boone et al. 2002). Ackerman and Iwama (2001) demonstrated increased hsp70 in two tissues during the course of an acute infection with pathogen Vibrio anguillarum, and it was thought that the observed concurrent Cortisol increase could be having such a dampening effect on hsp70 levels even though they were seen to increase. In using a Cortisol antagonist, it was expected that the effects of Cortisol increase would be mitigated, and a greater hsp70 response would be observed. However the opposite was found; there was virtually no effect on head kidney hsp70. There was a very small and transient increase in the first four days of infection in fish receiving the blocker but it differed from controls at only 5 d post-challenge. At this time though, the level did not differ from initial values for this same group. Liver hsp70 decreased two days post challenge in fish receiving the blocker and were lower than controls until day 6 at which time they did not differ from any other group. This response was unexpected and suggests that Cortisol may be a required component of the hsp70 increase observed in an earlier study (Ackerman and Iwama 2001). An alternative possibility is that during the period when levels decreased, hsp70 turnover may have been greater than production. However, this is speculative as protein turnover was not measured. The decrease in plasma protein levels at 4 d post-challenge was similar to that seen by Ackerman and Iwama (2001) where the greatest level of mortality corresponded to a decrease in plasma protein, likely due to haemodilution. This is further supported by the concurrent decrease in erythrocyte numbers and changes in haematocrit and haemoglobin. While the study provided unexpected and somewhat inconclusive results with respect to dynamics of the hsp70 response, it did yield some interesting information on use of RU-486 in immunological studies in fish. Other researchers found that RU-486 had no effect on glucose metabolism (Vijayan et al. 1994) and the present study provided similar results. However, where others reported that RU-486 had no effect on plasma Cortisol levels, this study showed a significant plasma Cortisol increase in fish implanted with the analogue versus sham controls (Figure 4-5) in the absence of challenge. Fish receiving the blocker plus disease challenge had an even higher plasma Cortisol concentration. While the physiological effects of Cortisol may be blocked, Cortisol concentration increased in circulation to levels generally considered indicative of a physiological stress response. Either RU-486 or the carrier may 108 have resulted in release of Cortisol into circulation, or its metabolic breakdown was slowed such that it built up in the plasma. Regardless, this is in contrast to what has been shown previously. Two early immunological responses to a stressor are a change in the proportions of circulating blood cells and an increase in lysozyme, a bacteriolytic enzyme produced by the macrophage/monocytes. Lysozyme levels in RU-486 challenged fish differed from controls at only 24 h post-challenge and there was a great deal of variation in both groups that received the analogue hormone. There was an effect on leucocyte populations in both RU-486 control and challenged groups. At 6 d post-challenge there were fewer total leucocytes in these two groups compared to sham controls. Within the leucocyte population, there was a decrease in lymphocytes and neutrophils between 4 and 6 days post injection and in both cases this differed from controls. While research has shown that there is a decrease in the ability of the anterior kidney to generate specific antibody producing ceils 4 h after an acute physical stress (Maule et al. 1989) several studies have shown, increased circulating levels of other leucocytes. Following stress (Tomasso et al. 1983; Angelidis et al. 1987; Ellsaesser et al. 1987;) inhibition of neutrophil apoptosis has been observed with Cortisol application (Weyts et al. 1998a; Weyts et al. 1998b) suggesting that although certain components of the acquired immune system may be downregulated with stress, certain non-specific immune components are up-regulated. If RU-486 is held to block effects of Cortisol at a physiological level, it obviously does not do so at the lymphocyte population level where results were similar to what one would observe in an animal under a normal physiological stress or a Cortisol implantation study. Although not statistically significant, difference in mortality between the two infected groups showed a tendency towards increased mortality in fish that challenged in combination with the blocker. One probable explanation for differences in controls between this and the earlier challenge study is that the bacterial dose that was aimed for was not achieved. This is supported by the fact that mortality in the current study was lower in control fish than previously. Although results were somewhat less clear than earlier experiments it is obvious that RU-486, although reported to block the actions of Cortisol but not its production (Reddy et al, 1995), either has detrimental health effects or does not block effects of Cortisol from an immunological standpoint, there may be non-genomic effects of Cortisol that have not been considered. RU-486 is a steroid receptor blocker that, while it does block the glucocorticoid receptor, is not specific for this receptor and this is an equal possibility from where the immune effects may stem. 109 Fish challenged in the presence of the Cortisol blocker had a significantly reduced plasma protein level and lower levels of stress protein in liver and head kidney tissues. They also had a higher (although not statistically) mortality rate. Although Cortisol is understood to have negative effects on the immune system, it appears that blocking its effects may be equally detrimental to the outcome of an infective process. While the study did not conclusively answer the question of whether or not Cortisol had a significant influence on stress protein levels during an acute bacterial infection, it did highlight some potential problems with the use of the Cortisol blocker RU-486 for immunological studies in fish. 110 References Ackerman, P. A., R. B. Forsyth, C. F. Mazur and G. Iwama. 2000. Stress hormones and the cellular stress response in salmonids. Fish Physiology and Biochemistry 23: 327-336. Balfry, S. K., M. Shariff and G. K. Iwama. 1997. Strain differences in non-specific immunity of tilapia Oreochromis niloticus following disease challenge with Vibrio parahaemolyticus. Diseases of Aquatic Organisms 30: 77-80. Basu, N., T. Nakano, E. G. Grau and G. Iwama. 2001. The effects of Cortisol on heat shock protein 70 levels in two fish species. General and Comparative Endocrinology 124: 97-105. Boone, A. A. and M. M. Vijayan. 2002. Glucocorticoid mediated attenuation of the hsp-70 response in trout hepatocytes involves the. proteosome. American Journal of Physiology Regulatory and Integrative Comparative Physiology 283: R680-R687. Colombo, L, A. D. Pickering, P. Belvedere and C. B. Schreck. 1989. Stress inducing factors and stress reaction in aquaculture. Aquaculture Europe '89 - Business Joins Science, Bordeaux, France, European Aquaculture Society. Forsyth, R. B., E. P. M. Candido, S. L. Babich and G. K. Iwama. 1997. Stress protein expression in coho salmon with Bacterial Kidney Disease. Journal of Aquatic Animal Health 9: 18-25. Gamperl, A. K., M. M. Vijayan and R. G. Boutilier. 1994. Experimental control of stress hormone levels in fishes: techniques and applications. Reviews in Fish Biology and Fisheries 4: 215-255. Houston, A. H. 1990. Blood and circulation. Methods for fish biology. C. B. Schreck and P. B. Moyle. Bethesda, Maryland, American Fisheries Society: 273-334. Laidley, C. W., P. T. K. Woo and J. F. Leatherland. 1988. The stress response of rainbow trout to experimental infection with the blood parasite Cryptobia salmositica Katz, 1951. Journal of Fish Biology 32: 253-261. Litwack, G. 1955. Photometric determination of lysozyme activity. Proceedings for the Society for Experimental Biology and Medicine 89: 401-403. Marx, J. 1995. How the glucocorticoids suppress immunity. Science 270: 232-233. Maule, A. G., R. Schrock, C. Slater, M. S. Fitzpatrick and C. B. Schreck. 1996. Immune and endocrine responses of adult chinook salmon during freshwater immigration and sexual maturation. Fish and Shellfish Immunology 6: 221-233. Maule, A. G., R. A. Tripp, S. L. Kaattari and C. B. Schreck. 1989. Stress alters immune function and disease resistance in chinook salmon (Oncorhynchus tshawytscha). Journal of Endocrinology 120: 135-142. Mommsen, T. P., M. M. Vijayan and T. W. Moon. 1999. Cortisol in teleosts: dynamics, mechanisms of action, and metabolic regulation. Reviews in Fish Biology and Fisheries 9: 211-268. Munck, A., P. M. Guyre and J. Holbrook. 1984. Physiological functions of glucocorticoids in stress and their relationship to pharmacological actions. Endocrine Reviews 5: 25-44. 111 Pegg, J. R., S. K. Balfry, L. Gordon, J. R. Roome and G. K. Iwama. 1995. Stress, immune function and disease resistance in juvenile salmonids. Bulletin of the Aquaculture Association of Canada 95: 28-35. Pottinger, T. G. 1990. The effect of stress and exogenous Cortisol on receptor-like binding of Cortisol in the liver of rainbow trout, Oncorhynchus mykiss. General and Comparative Endocrinology 78: 194-203. Pratt, W. B. 1993. The role of heat shock proteins in regulating the function, folding and trafficking of the glucocorticoid receptor. Journal of Biological Chemistry 268: 21455-21458. Reddy, P. K., M. M. Vijayan, J. F. Leatherland and T. W. Moon. 1995. Does RU486 modify hormonal responses to handling stressor and Cortisol treatment in fed and fasted rainbow trout? Journal of Fish Biology 46: 341-359. Smith, P. K., R. I. Krohn, G. T. Hermanson, A. K. Mallia, F. H. Gartenr, M. D. Provenzano, E. K. Fujimoto, N. M. Goeke, B. J. Olson and D. C. Klenk. 1985. Measurement of protein using bicinchoninic acid. Analytical Biochemistry 150: 76-85. Specker, J. L, D. M. Portesi, S. C. Cornell and P. A. Veillette. 1994. Methodology for implanting Cortisol in Atlantic salmon and effects of chronically elevated Cortisol on osmoregulatory physiology. Aquaculture 121: 181-193. Trinder, P. 1969. Determination of glucose in blood using glucose oxidase with an alternative oxygen acceptor. Annals of Clinical Biochemistry 6: 24-27. Tripp, R. A., A. G. Maule, C. B. Schreck and S. L. Kaattari. 1987. Cortisol mediated suppression of salmonid lymphocyte responses in vitro. Developmental and Comparative Immunology 11: 565-576. Vijayan, M. M., C. Pereira, E. G. Grau and G. K. Iwama. 1997. Metabolic responses associated with confinement stress in tilapia: the role of Cortisol. Comparative Biochemistry and Physiology 116C: 89-95. Vijayan, M. M., P. K. Reddy, J. F. Leatherland and T. W. Moon. 1994. The effects of Cortisol on hepatocyte metabolism in rainbow trout: a study using the steroid analogue RU486. General and Comparative Endocrinology 96: 75-84. Wendelaar Bonga, S. E. 1997. The stress response in fish. Physiological Reviews 77: 591-625. Weyts, F. A. A., G. Flik, J. H. W. M. Rombout and B. M. L. Verburg-van Kemenade. 1998. Cortisol induces apoptosis in activated B cells, not in other lymphoid cells of the common carp, Cyprinus carpio L. Developmental and Comparative Immunology 22: 551-562. Weyts, F. A. A., G. Flik and B. M. L. Verburg-van Kemenade. 1998. Cortisol inhibits apoptosis in carp neutrophilic granulocytes. Developmental and Comparative Immunology 22: 563-572. Yin, Z., T. J. Lam and Y. M. Sin. 1995. The effects of crowding stress on the non-specific immune response in fancy carp (Cyprinus carpio L.). Fish and Shellfish Immunology 5: 519-529. 112 Section IV. Cell culture studies on the cellular stress response to immunological challenge during exposure to physiological levels of Cortisol Hans Selye (1967) said "You can never learn what a mouse is like by carefully examining each of its cells separately under the electron microscope any more than you could appreciate the beauty of a cathedral by chemical analysis of each stone which went into its construction". Physiological and immunological systems are closely tied and there are inherent problems in attempting to pull them apart to study one or the other, especially at the whole animal level. Intuitively, there are many systems functioning during any process and it is difficult, at best, to draw solid conclusions when so many levels of organization are at work. In order to fully understand an organism or the processes that take place within it at any one point in time, we must look at it at all levels of organization. Previous experiments in this thesis focused on responses of fish at a whole animal level to immune challenge, lethal and non-lethal. An attempt was made to remove the actions of Cortisol in the whole animal using an antagonistic blocker and while physiological effects of Cortisol may have been blocked, it was not as clear as to what effects RU-486 had on other factors such as disease susceptibility. Cell culture studies provide the ability to study isolated cells of interest and examine their response in the presence and absence of natural physiological products. This final study was undertaken to explore effects of an immunological challenge and the stress hormone Cortisol on hsp70 responses at the cellular level in two tissues that have been examined throughout this thesis. 113 CHAPTER 5 The cellular stress responses of primary cultured rainbow trout hepatocytes and a macrophage-like Atlantic salmon cell line (SHK-1) to experimental immunological challenge. Introduction The immune system generally functions quietly in the background, providing silent protection against bacterial invaders. It has been studied extensively and we are just beginning to tease apart some of the connections between this and other systems. Historically, physiological and immunological studies have been quite separate. Only in the last 60 years have researchers begun to examine effects of physiological stressors on the immune system and it has been repeatedly demonstrated that stress generally has detrimental effects on disease resistance. However, it is becoming increasingly apparent that we have merely scratched the surface of these relationships. While stress seems to have negative effects on many components of the fish immune system (Mockef al. 1990; Fevolden et al. 1992; Maule et al. 1996) the mechanisms are poorly understood and few studies have described effects of infection from a physiological perspective. Non-specific defence mechanisms of fish have been extensively studied (Dalmo et al. 1997) and it is well known that the head kidney is a tissue rich in haematopoietic cells. Self and non-self substances are processed in the head kidney and several studies have examined translocation and processing of a variety of substances in this region (Bogwald et al. 1996; Espenes et al. 1996; Chen et al. 1998; Stensvag et al. 1999). A major toxic factor in a Gram-negative bacterial infection is lipopolysaccharide (LPS). It has been demonstrated that bacterial LPS is taken up in both the head kidney and liver (Dalmo et al. 1996; Dalmo et al. 1998; Stensvag et al. 1999) and although this endotoxin is responsible for mammalian septic shock during an infection, fish are extremely resistant to it and the reasons for this are unknown. The heat shock proteins (hsp) are a class of highly conserved and inducible proteins (Lindquist 1988) first observed as chromosomal puffing initiated by a thermal stress (Ritossa 1962) but their induction has been demonstrated in response to a variety of stressors and environmental contaminants other than thermal shock (Sanders 1993). These proteins play important roles in maintenance of homeostasis through protein transport, folding, and degradation processes (Morimoto et al. 1990), but additional functions of hsps are still being elucidated. They are thought to be involved in adaptation of cells such that a non-lethal pre-treatment induces synthesis of proteins that protect the cell from subsequent shock that may 114 otherwise be lethal (Ciavarra et al. 1990). They have been examined closely for potential use as biomarkers for environmental monitoring (Sanders 1990; Sanders 1993), but as increasingly more events are demonstrated to induce the response, their suitability for identifying specific environmental stressors becomes questionable. While early studies of the hsp response centered on their increase relative to heat and chemical stressors, much of the present focus has shifted to their role in the immune system. Studies with mammals have demonstrated an important role for hsp in the pathology of some mammalian inflammatory diseases (Schett etal. 1998) in which there is strong induction of hsp (reviewed by Jaquier-Sarlin et al. 1994) possibly as a protective function against auto-oxidative stress brought about by activated immunocytes. In mammals, hsp70 is found at higher levels in the phagocytic monocyte/macrophage line of cells than in any other cell type tested and functions ranging from self/non-self recognition to antigen presentation have been attributed to this family of proteins (reviewed by Bachelet et al. 1998). Bacterial infection resulting in activation of phagocytes and granulocytes leads to an increase in bacterial killing activity through mechanisms such as the production of lysozyme, cationic proteins, and reactive oxygen species (ROS i.e. hydrogen peroxide, hydroxyl radical, etc) brought about by respiratory burst activity. These same molecules are also damaging to components of the host cell and it has been postulated that one function of hsp production in these cells is to protect against self damage due to auto-oxidation (Bachelet ef al. 1998). While the fish immune response has been extensively studied at both the whole animal and cellular levels, there is little information on the cellular stress response to immunological challenge. Forsyth ef al (1997) were the first to look at hsp70 production in response to a bacterial infection in fish. These authors found an increase in tissue levels of hsp70 that were closely related to foci of Renibacterium salmoninarum infection. However these changes occurred during advanced disease progression when strong clinical signs were visible. Ackerman ef al (2001) subsequently investigated physiological responses to an acute bacterial infection (Vibrio anguillarum) and found a similar hsp70 response, although it occurred at a much earlier point in disease progression and prior to any clinical signs of infection. Subsequent experiments examined the role of bacterial lipopolysaccharide (LPS) and killed bacterial cells in generating these same responses and the role of Cortisol during an infection (Chapters 3 & 4). This study was undertaken to examine the previously recorded responses at a cellular level and determine if they could be reproduced in the primary cell types that make up the two tissues under examination, head kidney and liver. 115 Methods and Materials Cell Culture - A macrophage-like cell line (SHK-1) from Atlantic salmon (Salmo salar) was provided by the National Research Council Institute for Marine Biosciences (originally obtained from B.H. Dannevig, Norwegian College of Veterinary Medicine, Oslo Norway) and was maintained according to a modification of the method outlined by (Dannevig et al. 1995). Briefly, cells were grown in Liebovitz's L-15 cell culture medium supplemented with FCS (5% v/v) and gentamycin (50 pg/mL) using Falcon Primaria tissue culture flasks. Cells were harvested from culture flasks as follows. Media was poured off and flasks were rinsed gently with 10 mL Dulbeco's Ca + and Mg 2 + free PBS. Cells were detached from plates by adding 3 mL trypsin (0.05% w/v) EDTA (0.02% w/v) in Dulbeco's PBS and washed 3 x by centrifugation (5 min @ 500 x g in 10 mL L-15 media containing no antibiotics). Cells were resuspended to a final concentration of 7 x 10 5 cells/mL in Liebovitz's L-15 medium with FCS (5% w/v) based on counts performed in a haemocytometer using Trypan blue stain to differentiate live cells, sterile 24 well tissue culture treated polystyrene plates (Corning Costar 3524) were seeded with 1 mL cell suspension for a final density of 7 x 10 5 cells per well. Plates were placed in a moist chamber in an incubator at 17°C and allowed to adhere for 24 h by which time they had formed a confluent monolayer on the well bottom. Primary hepatocytes from three rainbow trout (approximately 200 g each) were isolated and cultured according to Mommsen et al. (1994). Cells were resuspended to a density of 7.5 x 10 5 cells/mL and 500 pL/well of this suspension was plated onto sterile 24 well tissue culture treated polystyrene plates (Corning Costar 3524). Plates were placed in moist chambers in an incubator at 12°C and cells were allowed to settle and adhere for 24 h prior to experimental challenges. Cell viability was evaluated by Trypan blue stain (0.2%; 5 to 15 min) exclusion. Values higher than 90% were considered adequate. Treatments - The primary isolate (Pacific Biological Station, Nanaimo, B.C., isolate number 98055) of V. anguillarum (serotype 02) was obtained from wild chum salmon (O. keta) that had died from natural exposure to the pathogen. Twenty hour V. anguillarum cells were harvested from tryptic soy agar (TSA, supplemented with 1.5% NaCI) plates, transferred to sterile peptone-saline (P-S: 0.2 % peptone, 1.5% NaCI) and vortexed to make a suspension. The concentration of the V. anguillarum suspension was estimated from absorbance measurements made at 540 nm (1 O D 5 4 0 estimated to contain 109 cells/mL). The suspension was diluted to approximately 10 7cfu/mL and 10 uL of this suspension was added to each well receiving this treatment. Actual doses were determined by drop plate counts. SHK-1 cell 116 culture wells received an actual dose of 1.67 x 10 6 cfu/mL and hepatocyte cultures received 1.58 x 10 5 cfu/mL. Both SHK-1 and hepatocyte cell culture experiments tested the same following 11 treatments: 1) Sterile physiological saline at pH 7.2 2) E.coli LPS at 200 pg/mL final concentration 3) V. anguillarum LPS at 200 pg/mL final concentration 4) Killed V. anguillarum at 106 cells/mL final concentration 5) Live V. anguillarum at 105 cells/mL final concentration 6) Sterile saline + 300 ng/mL Cortisol final concentration 7) E.coli LPS at 200 pg/mL + 300 ng/mL Cortisol final concentration 8) V. anguillarum LPS at 200 pg/mL + 300 ng/mL Cortisol final concentration 9) Killed V. anguillarum at 106 cells/mL + 300 ng/mL Cortisol final concentration 10) Live V. anguillarum at 10s cells/mL + 300 ng/mL Cortisol final concentration 11) 30°C Heat shock - to ensure that cells were capable of mounting an hsp 70 response A bacterin was prepared according to Stolen et al. (1990) from the primary isolate (Pacific Biological Station, Nanaimo, B.C., isolate number 98055) of V. anguillarum. Briefly, 20 h V. anguillarum cells were harvested from tryptic soy agar (TSA, supplemented with 1.5% NaCl) plates, transferred to sterile peptone-saline (P-S: 0.2 % peptone, 1.5% NaCl) and gently vortexed to make a suspension and avoid clumping of cells. The suspension was then centrifuged until the supernatant was clear (1600 x g, 4°C for 25 min). The supernatant was discarded and the cell pack resuspended to a 0 .1% suspension in 0.4% formalin saline. This was incubated overnight at 4°C under gentle agitation. The solution was washed 3 x by centrifugation in phosphate buffered saline (PBS) and resuspended each time in roughly 10 x the cell pack volume. The final suspension was estimated at 10 8 cells/mL (1 OD54onm = 10 9 cells/mL) and each well was exposed to a final concentration estimated at 10 6 cells/mL. LPS from E. coli (Serotype 026:B6, Sigma L3755) and V. anguillarum (Sadovskaya et al. 1996) were suspended in physiological saline (pH 7.2). Each well being treated with LPS was exposed to a final concentration of 200 pg/mL. Cortisol was dissolved in sterile physiological saline (pH 7.2) and wells receiving this treatment were exposed to a final concentration of 300 ng/mL. Cortisol exposure concentration (300 ng/mL) was chosen based on average levels seen in fish exposed to an initial challenge experiment (Ackerman et al. 2001). Two LPS challenges were carried out as earlier whole animal experiments were 117 performed using E. coli LPS and the cell studies presented an opportunity to examine and compare the responses to both V. anguillarum and E. coli LPS. Samples were collected to examine differences in hsp70, lysozyme production, and LDH. To ensure that cells were capable of mounting a hsp70 response, a heat shock was performed. Plates were taken from the cool incubator and placed in a moist chamber in a high temperature incubator at 30°C for 1 h. Cells were then returned to the moist chamber in the cool incubator. Cell Harvesting - SHK-1 cells were harvested from plates at 4 and 24 h as follows: Dulbeco's PBS without Ca + or Mg 2 + (100 uL) was added to each well and gently swirled to clean cells and remove dead or unattached cells. This was pipetted off gently, 100 pL of Trypsin-EDTA solution was added to each well, plates were swirled gently to detach cells. Dulbeco's PBS containing Ca + and Mg 2 + (200 pL) was added to each well and cells and solution were removed by pipette and transferred to 1.5 mL Eppendorf tubes. Wells were then rinsed once with 200 uL Dulbeco's PBS containing Ca + and Mg 2 + which was removed and added to the Eppendorf tube. Tubes were centrifuged at 500 x g for 5 min and the supernatant was removed and transferred to a separate tube. Hepatocytes were harvested at 4 and 24 h as follows: plates were chilled on ice and a pipettor set to 400 pL was used to gently flush cells off the well surface. Medium was drawn up the pipettor and expelled gently across the bottom of the well. Flushing was repeated 5 x in each well to cover the entire well surface and ensure all cells had been dislodged. The suspension was then collected into Eppendorf tubes and centrifuged for 30 sec at 6500 rpm in a microcentrifuge. The supernatant was removed and saved for LDH analysis. Pellets were rinsed once gently with 50 pL sterile PBS to remove any residual L-15 medium. Analytical Procedures - Preparation of samples, and determination of hsp70 was carried out by a modification of Forsyth et al. (1997) and reported as units relative to a positive arsenite induced control. Briefly, frozen cell pellets were resuspended in ice cold lysis buffer (50 mM Tris, 5 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 1 mM Pepstatin A, 1 mM Leupeptin and 0.015 mM Aprotinin), sonicated (20 MHz) on ice, vortexed, and placed back on ice. Aliquots (10 pL) of each sample were frozen immediately on dry ice and stored at -70oC. The hsp70 antibody used was prepared as outlined in Forsyth et al. (1997). The supernatant was assayed for LDH activity according to Bergmeyer (1974). Lysozyme activity in the supernatant from the SKH-1 cell line was determined using a modification of Litwack's (1955) method (Maule et al. 1996). The method was modified for use on a microplate reader and used 10 mL of supernatant (or hen egg white lysozyme (HEWL) standard), and 250 mL of 0.025% 118 w/v Micrococcus lysodeikticus suspension in 0.06 M phosphate buffer (pH 6.2). The decrease in optical density over 20 min of incubation at 25°C is reported here as pg/mL equivalent of HEWL activity. Statistical Analysis - Data are presented as means ± 1 standard error (SE). When analysis of variance indicated significant differences and data passed the normality test, the Student-Newman-Keuls all pairwise multiple comparison test was used to identify significantly different means (P < 0.05). Where data failed the normality test, Dunn's method of all pairwise multiple comparison was used for this purpose. The level of significance in all tests was P < 0.05. Results Lysozyme - Lysozyme activity in SHK-1 cell culture supernatant increased between 4 and 24 h in all treatment groups except controls, but this increase was only significant in cells exposed to live V. anguillarum cells or those exposed to E. coli LPS with Cortisol. Cultures exposed to V. anguillarum + Cortisol or E. coli LPS alone did not have a similar increase (Figure 5-1). Four hours post exposure, the only significant difference between any treatment groups was between E. coli LPS + Cortisol and killed V. anguillarum + Cortisol (Figure 5-2). Application of Cortisol did not appear to have any effect on lysozyme activity levels among individual treatment pairs (with vs. without Cortisol) except E. coli LPS where activity was observed to be statistically lower, but only marginally so. At 24 h post exposure, the bacterin + Cortisol had a greater supernatant lysozyme activity compared to saline + Cortisol controls (Figure 5-2). Hsp70 - A 30°C heat stress (SHK-1 +15°C & hepatocyte +18°C) was sufficient in both studies to result in a significant increase in cellular hsp70 indicating that both cultures were capable of mounting a hsp70 response. While hepatocytes had a significant increase in hsp70 at 4 h post heat treatment (Figure 5-6) this was not seen in SHK-1 cells (Figure 5-4) suggesting that perhaps these cells were less sensitive to cellular stress. At 24 h post exposure only saline + Cortisol and live V. anguillarum + Cortisol treatment groups were significantly different from controls in SHK-1 cells. Although there was a slight decrease in hsp70 in cells exposed to the live pathogen with Cortisol added compared to pathogen alone, there was no statistical difference between them. When there was no immunological stressor applied there was a significant difference between SHK-1 cells with and without Cortisol (i.e. saline compared to saline + Cortisol controls). No differences were observed between any treatments in SHK-1 cells at 4 h. 119 At 4 h post exposure, hsp70 levels in hepatocytes were significantly lower in cells treated with live V. anguillarum + Cortisol compared to all other treatment groups and, excluding heat shock, levels were highest in cells exposed to E. coli LPS both with and without Cortisol (Figure 5-6). At 24 h, E. coli LPS, with or without Cortisol, resulted in a significantly greater hsp70 level compared with cells treated with killed or live V. anguillarum alone. Differences within treatments and between time periods were seen for both cell cultures. There were significantly lower levels of hsp70 in SHK-1 cells receiving E. coli LPS alone, V. anguillarum LPS with Cortisol, killed V. anguillarum with Cortisol, and live V. anguillarum with Cortisol (Figure 5-3). This trend was similar for all remaining treatments including both sets of controls. Only heat shock resulted in an increase in hsp70 levels between 4 and 24 h in these cells. Hepatocytes, in contrast, had an increase in hsp70 between 4 and 24 h in cultures receiving E. coli LPS and V. anguillarum LPS alone, and for every treatment receiving Cortisol. Heat shock also resulted in a significant increase in hsp70 between 4 and 24 h (Figure 5-5). LDH - At 4 h SHK-1 culture supernatant LDH activity was significantly increased in cultures exposed to V. anguillarum + Cortisol and V. anguillarum LPS. At 24 h post exposure, LDH activity in supernatant from those cells exposed to the live pathogen, both in the presence and absence of Cortisol, was greater than any other treatment group, but not significantly different from .each other. These levels were significantly greater than levels observed in heat shocked cells. Differences between samples at 4 and 24 h within treatments were only observed for live V. anguillarum and V. anguillarum with Cortisol (Table 5-1). Hepatocyte culture supernatant LDH activity at 4 h was significantly greater in cultures exposed to E. coli LPS, killed V. anguillarum, and E.coli LPS + Cortisol compared with controls and most other treatment groups (Table 5-1). LDH was significantly greater in heat shocked cultures than in all other groups at both 4 and 24 h post exposure. At 24 h, £. coli LPS and V. anguillarum LPS + Cortisol yielded significantly higher LDH activities than all other groups except for heat shocked cells. Differences within treatments between sampling times were observed for cells exposed to £. coli LPS (increased) and killed V. anguillarum (decreased) alone. Within treatments exposed to Cortisol, there was a general decreasing trend for all but the groups exposed to V. anguillarum + Cortisol or heat shock, which had an increase in LDH activity. Cultures exposed to both E. coli + Cortisol or V. anguillarum + Cortisol had a significant decrease in LDH between 4 and 24 h. 120 2.5 i 2.0 1.5 Cortisol absent I X :> 1.0 0.5 0.0 Cortisol present X heat shock only co cz ro CO J Z SZ stf s* s* CM CO CM CO CL CO cz _ l L ro — _ l CT) O o O LU co LU sz st 00 CL I CD C ro > sz «* CM CO CL CT c CO CJ) c ro > X J CO XT CM CD C ro > "D CD CD C ro co > CM CT C ID CO > > * n J Z st st CM o to O — CO o TZ o O + O CO + cz CO — c ro CO ro CO o to i_ o u + CO CL Q to LU CM O CO O CJ CO CL o o LU J Z J Z x t XT CM o to O to o r o o + o CO + CL CO _ l L CD _ l C CD ro CZ > ro > J Z J Z J Z J Z st st s t st CM CM o O to o CO O r CO 'TZ to o '•c o •c o o o o + o + CJ CT + CT + C C CT ro c ro c > ro > ro T J > CD > 0 T J > CO _C0 > 5 J Z to o J Z to ro CD X st CM O o ra co X Figure 5-1. Lysozyme activity in SHK-1 cells at 4 and 24 h following challenge in the presence or absence of Cortisol (300 ng/mL). Means are presented ± SE. Asterisks denote statistical differences between sampling periods within treatment between 4 and 24 h only (P < 0.05). 121 2.5 -i 2.0 1.5 1.0 0.5 H 0.0 4 h post exposure ab ab ab ab ab CD c "CD CO T -C "o CO 8 + CO ro CO ab ab 3b ab co L L O O UJ J Z J Z "5 co "o co Q . CO •fe! _ J •c o CO q o c o + CVS + co > CO C L D _ _ J _1 1ZZ CO O c o rc LU > CO c ro "O CD J Z o ' t . o o co c CD T 3 CD CD C CD > CD > CO c CD > CD > o o J Z CO ra CD X 24 h post exposure ab ab ab ab T x -1 J Z i J Z 1 J Z 1 J Z i J Z 1 J Z "tf •«* "<* «*• C N C M C M C M C M C M 0 o co "5 co "o c CO CO C L CO " ro _ J " t . _ I 'XL co o o CO O o O o c o + 0 + CD + CD LU CO > CO c C L C L "CD _l _ J CO — CO o cn 0 CD ai > T J CD CD > Figure 5-2. Lysozyme activity in SHK-1 cells at 4 and 24 h following challenge in the presence or absence of Cortisol (300 ng/mL). Means are presented ± SE. Letters denote statistical differences between treatments within each sampling time period (4 or 24 h) only. (P < 0.05). 122 250 -i Cortisol absent 200 150 H 100 4 50 X I I I I Cortisol present X XT CD C "ro CO - t CN CD C ro co co O O LU St S* ^ CM ^ CM CO Q. CO 0_ _l Q_ s t CD C CO JZ SZ SZ. st st st CNI ^ CN O CJ U J CD C ro -1 > T J > fj; CD T J ro = 0 > * I CO c c ro ro CD > CO c ro > CD > SZ J Z J Z J Z J Z J Z J Z J Z J Z s t s t - t s t • t s t s t s t s t CM CM CM ~o CM 7-i CM CO "o CO "o CO o \J CO "5 IS> o 'tr CO 't_ CO tr: CO 'TZ CO tr. CO o o 'tz: o tr. o r o tr. CJ o CJ o 0 o o o CJ o + o + o + CJ + CJ + o cu + CO + CO + CO + co + c CD n CO CL CO cz CO c CD Sali Salin col LI CL _ J "o _l CO c CL _1 CO ro > T J V.an eV.a V.an LU o CO cz T J > CO uJ > CD _CD _ i > > 2 _l Figure 5-3. Hsp70 in SHK-1 cell culture at 4 and 24 h following challenge in the presence and absence of Cortisol (300 ng/mL). Means are presented ± SE. Asterisks denote statistical differences between sampling periods within treatment between 4 and 24 h only (P < 0.05). 123 250 -i ^ - .200 co - 4 — ' 'rz =3 CD > 150 CD o Q. co 50 -I 4 h post exposure X X X X 24 h post exposure ab X ab X ab ab ab ab ab X b CD c " r o C O o co •c o o CD E ro co J Z ^r co Q. o o LU o CO 't. o o co 0_ o o LU •sr C O C L _ l cn cz CD > o CO n o o co c ro CD co CL _ l C D SZ CD r^ co c ro > CD > cn C co T J CD J Z O CO t. o o CO c ro > > J Z r^ j*: o o J Z CO "5 CD X J Z JZ J Z r^ C N CD c ro CO ^ •sf CM C M o CO 'tu. o o + CD C "ro co co C L O o LU > = CM O co r o o + CO C L O o LU ^r CM CO C L CO c CD > CM O CO '•c o o + 00 C L _ I co c ro > ^r C M C M C M C M C O c ro CD O CO iz o o + CO c ro T J CD CO c ro CD > o CO T Z o o + CO c ro CD > J Z r^ CM o o J Z CO CO CD X Figure 5-4. Hsp70 in SHK-1 cell culture at 4 and 24 h following challenge in the presence and absence of Cortisol (300 ng/mL). Means are presented ± SE. Letters denote statistical differences between treatments within each sampling time period (4 or 24 h) only (P < 0.05). 124 300 CO *z 250 13 CD | 200 0 Cortisol absent o Q. CO V / A 60 40 20 n l I n n Cortisol present * ft a n G ro CO st CM CD .cz "CTJ CO co fj_ o o LU CN CO C L o o LU CO C L co cr ro J Z J Z J Z J Z J Z J Z t t t t t t ™ CD ™ CD N o CO C D) C Q_ CD C CO 'S> rz CO = O> CD -Q CD TJ iv) = CD >*1 CO > CO c ro > CD > CD C CO CO XT CN "o S3 'XL o o + CD c ra CO J Z J Z st st CN O co O 'tr CO o t a O + o CO + C L CO _ J Q_ _ J o o o L U o L U o S3 "tr. o a + co C L _ I co c CO > J = J Z J Z J Z J Z J Z st st ^t st st CN o CN o CN Cj O co a co "o n co 'tr CO n CO sz 'tr o tr: o 'tr CO o o o o o o + o + o Hea + CO + CO + Hea 00 rz CO cr CO Q_ CO c CO c _ J > ro > CO CD T J > CD > cz 0) > CD ro > > iZ — _ J 2H CN zn 3 o JZ CO CD Figure 5-5. Hsp70 in hepatocyte primary cell culture at 4 and 24 h following challenge in the presence and absence of Cortisol (300 ng/mL). Means are presented ± SE. Asterisks denote statistical differences between sampling periods within treatment between 4 and 24 h only (P < 0.05). 125 300 -i 4 h post exposure co ' c Z S CD > ro cu s— o S-Q_ C O X 250 200 40 20 A a « a * J Z J Z J Z J Z J Z J Z T t f s t s t CD "o co o CO "o C CO C L CO C L CO ra t _ l r _ l C O o •jzz o CO o o O CJ c CJ + CJ + CD + cu cz ui C O > C O Q . D _ " r o _ l CO — CO o c CJ ro L U > c sz sz sz sz sz •<T X T - T T T T T 24 h post exposure ae be X ae be a X E CO £ CO ro -TZ ro T J CD 8 > + f CO -1 c ro T J CD rz o o o o J Z co ro CD CO X c ro cu > J Z J Z J Z J Z J Z J Z ST s t s t s t s t CN C N C N CN CN C N 0 ~ o CO "o c o "o c CO CL CO C L CO ro 'tz _ l tz _ l 'rz CO o — o CO o CJ o o c o + o + ro + CD L U C O > CO rz D _ D _ "ro _ _J CO CO c 1 ra L U > t t t C N C N C N CO c ra T J CD CO c ro > CD > O CO 'rz o o CO c ra > T J CD CO rz CD CD > J Z *tf CN o c J Z cn ro CD x Figure 5-6. Hsp70 in hepatocyte primary cell culture at 4 and 24 h following challenge in the presence and absence of Cortisol (300 ng/mL). Means are presented ± SE. Letters denote statistical differences between treatments within each sampling period (4 or 24 h) only (P < 0.05). 126 Table 5-1. LDH Activity in SHK-1 and hepatocyte cultures following experimental exposure to immunological challenge in the absence and presence of exogenous Cortisol (300 ng/mL). Superscript letters denote differences (± SE) between treatments within sampling time periods (4 or 24 h), numbers indicate differences (± SE) within treatment between 4 and 24 h sampling times (P<0.05, n=10). SHK-1 cell line Hours post exposure 4 24 Saline 0.0006 ± 0.00023 0.00051 0.0001a Saline + 300ng/mL Cortisol 0.0003 ± 0.000073 0.0007 ± 0.0002a E.coli LPS 0.0003 ±0.0001 a 0.0006 ± 0.0001a E.coli LPS at 200 pg/mL + 300ng/mL Cortisol 0.0003 ± 0.00008a 0.0005 ± 0.00013 V. anguillarum LPS 0.001 ± 0.0003DC 0.0006 ± 0.00023 V. anguillarum LPS at 200 pg/mL + 300ng/mL Cortisol 0.0008 ± 0.0003ac 0.0009 ± 0.00023 Killed V. anguillarum at 10 b cells/mL 0.0005 ± 0.00023 0.0005 ± 0.000093 Killed V. anguillarum at 10 b cells/mL + 300ng/mL Cortisol 0.0006 ± 0.0001a 0.001 ± 0.00033 Live V. anguillarum at 10 s cells/mL ••0.0007 ± 0.0003ac *0.02 ± 0.0008° Live V. anguillarum at 10 a cells/mL + 300ng/mL Cortisol '0.002 ± 0.0003d 20.02 ± 0.0009° 30°C heat shock 0.001 ± 0.0002°° 0.001 ± 0.000093 Hepatocyte primary culture Hours post exposure 4 24 Saline 0.002 ± 0.0006a 0.002 ± 0.00063 Saline + 300ng/mL Cortisol 0.008 ± 0.001D a 0.004 ± 0.0023 E.coli LPS '0.004 ± 0.0009ac z0.010 ± 0.002° E.coli LPS at 200 pg/mL + 300ng/mL Cortisol '0.008 ±0.001° *0.003 ± 0.0013 V. anguillarum LPS 0.003 ± 0.0008ac 0.003 ± 0.002a V. anguillarum LPS at 200 pg/mL + 300ng/mL Cortisol 10.005 ± 0.002 a c a ^0.01 ±0.001° Killed V. anguillarum at 10 b cells/mL '0.006 ± 0.001D C ^0.002 ± 0.00083 Killed V. anguillarum at 10 b cells/mL + 300ng/mL Cortisol 0.004 ± 0.0010ac 0.002 ± 0.00053 Live V. anguillarum at 10 a cells/mL 0.005 ± 0.002a c 0.004 ± 0.00073 Live V. anguillarum at 10 s cells/mL + 300ng/mL Cortisol '0.005 ± 0.0009ac *0.002 ± 0.00053 30°C Heat shock 0.01 ± 0.002e 0.02 ± 0.002° 127 Discussion In the current study, we were interested in learning if the responses in two tissues, head kidney and liver, to an immunological challenge provided by a Gram-negative pathogen would be observed in isolated cells of those tissues. We also investigated effects that Cortisol at a biologically relevant stress level had on the hsp70 response in those cells during such a challenge. Providing insights into these responses may help clarify processes that the system undergoes during either a disease process or an immunization and may assist in development of improved vaccines or therapeutants. Lysozyme is an antibacterial enzyme that is often measured as an indirect evaluation of non-specific immune function. It is a secretory product of macrophage/monocytes and acts by disrupting the peptidoglycan layer of bacterial cell walls, either alone or in conjunction with other antimicrobial molecules such as complement. Plasma lysozyme has been observed to increase as soon as 10 min following a physiological stress (Demers ef al. 1997) and its increase has been suggested to be a compensatory adaptation to bolster immune resistance during a Cortisol increase that would normally be regarded as immunosuppressive. The head kidney is rich in cells that produce lysozyme and SHK-1 cells are derived from this tissue. Lysozyme activity in SHK-1 cell culture supernatant increased between 4 and 24 h in most treatment groups except controls indicating that all treatments, including Cortisol alone, had a stimulatory effect on the release of this immunologically active enzyme. This indicates that all treatments caused a stress response, although to varying degrees. However, this increase was only statistically significant in cells exposed to the live pathogen and £. coli LPS in conjunction with Cortisol. It is somewhat surprising that V. anguillarum LPS did not cause a similar result. Cortisol does appear to have diminished the difference somewhat, but again, this was not significant. Fevolden et al (1992) reported a higher level of lysozyme activity in fish that responded to stress with a large increase in Cortisol (Fevolden ef al. 1992). Cortisol alone was insufficient to generate a statistically significant increase in lysozyme activity at the cellular level. However, cells that were exposed to E. coli LPS in conjunction with Cortisol had a greater change in lysozyme activity between 4 and 24 h than those exposed to E. coli LPS alone. It is not known why cells did not respond in a similar manner to the V. anguillarum LPS. The toxic component of LPS is highly conserved and studies investigating its effects on immunomodulation have demonstrated that it is capable of enhancing macrophage phagocytic activity, migratory activity, and superoxide anion production both in vivo and in vitro (MacArthur ef al. 1985; Salati ef al. 1987; Solem ef al. 1995). In whole animal studies, 128 concentrations of LPS up to 355 mg/kg fish weight have been injected without observable adverse effects (Harbell 1972); fish appear to clear (and detoxify) it quickly. Even though the structure is highly conserved, it stands to reason that there will be differences between LPS from different bacteria and these differences will result in variations in responses at the cellular level. E. coli can colonize the salmonid intestine at warmer temperatures (>15°C), but it is generally assumed that infection occurs when fish pass through contaminated water sources and it is not normally a component of the fish intestinal tract (Del Rio-Rodriguez et al. 1997). If this is true, it is less likely that any long standing ecological relationship between E. coli and salmonids has occurred in the aquatic environment. It stands to reason then, that fish should react in a stronger manner towards an LPS that is "more foreign" than one that has been encountered through evolutionary history. This may be the reason that SHK-1 cells responded with a stronger lysozyme response to the E. coli LPS in the presence of Cortisol. Hsp70 levels are commonly measured as cellular indicators of stress in fish (Forsyth et al. 1997; Iwama et al. 1999; Ackerman et al. 2000; Nakano et al. 2002) but often these stressors are outside of normal biological limits that an organism may be expected to encounter. Both cell cultures were capable of mounting cellular stress responses as measured by hsp70. It was expected that responses to immunological challenge in cell cultures would mirror those observed in whole animal studies (Ackerman et al. 2001). However, where hsp70 levels in both liver and head kidney tissues increased in response to disease in whole animals, this was not mirrored in culture for either tissue. It was previously suggested that the increase in hsp70 in tissues from whole animals might be due in large part to cellular damage (Ackerman et al. 2001). LDH activity in culture supernatant is an often used indicator of cell viability and an increase is correlated with decreased cell viability. This is therefore, an accepted indicator of cellular damage. In the SHK-1 cell line, infected cultures produced the greatest LDH activity indicating that these cells suffered the greatest cellular damage. Yet these same cells contained the lowest levels of hsp70 where oniy the live pathogen in conjunction with Cortisol resulted in levels significantly different from controls. Fish hepatocytes are often used to study effects of environmental stressors on cellular physiology (Koban et al. 1987; Currie et al. 2000; Boone et al. 2002) and appear to be sensitive indicators of stress. However, to the best of our knowledge, to date there have been no studies involving effects of V. anguillarum using these cells or the SHK-1 line on hsp70 production in response to immunological challenge. While hepatocytes had a significant increase in hsp70 in response to heat shock at 4 h post heat treatment, this was not mirrored in SHK-1 cells, suggesting that perhaps the latter were less sensitive to cellular stress. Heat 129 shock resulted in significantly greater levels of hsp70 compared with any other treatment in both cell cultures at 24 h post exposure, but this was expected since it represents an extreme cellular insult. When exposed to immunological challenge, either in the presence or absence of Cortisol, there were response differences between the two cell types. No differences were observed between any treatment at 4 h in SHK-1 cells, and at 24 h post exposure there was still relatively little disparity. There was a clear difference in trends between the two cell cultures, although it appears that it may simply have been temporal. While there was no significant change in hsp70 at 4 h in SHK-1 cells, at 24 h in all treatment pairs (other than E. coli LPS) there appears to be a tendency towards lower levels of hsp70 in all treatments with Cortisol compared to those without. This was similar to the trend observed at 4 h in hepatocytes where, although again there were few statistical differences, there was a clear tendency showing reduced hsp70 in cells exposed to Cortisol. Interestingly, this trend was reversed in hepatocytes at 24 h. It has been reported that Cortisol may attenuate the hsp70 response in fish (Ackerman ef al. 2000; Basu ef al. 2001; Boone ef al. 2002). In the present study when there was no immunological stressor applied there was a significant difference between hepatocytes with and without Cortisol (i.e. saline compared to saline + Cortisol controls) suggesting that simply the presence of Cortisol in the media caused an increase in hsp70 production in these cells. Similar to what was found in whole animal studies, there are significant differences in tissue responses to immunological challenge. The liver is a detoxifying 'station' while the head kidney is a major site of immune cells and molecules, and this could underlie the differential response to pathogenic challenge. Bacterial LPS injected into fish as an adjuvant along with a bacterin has been suggested to boost the natural immune system and enhance acquired immunity to the bacterin (Anderson 1992; Ackerman ef al. 2000). The response to LPS included significant effects on hsp70 induction, but the relationship was not always clear. Even though other studies have failed to show physiological stress effects of LPS in salmonids (Harbell et al. 1979) it appears to have significant effects at the cellular level. While levels of hsp70 in hepatocytes increased in most treatments when Cortisol was applied, SHK-1 cells did not reflect this except in the case of E. coli LPS. In contrast, many of the levels instead had a decreasing trend. The data set demonstrates the complex nature of the effects of an immunological challenge, as well as the difficulty in fully understanding these when cells are removed from the natural system of the whole animal. This is the first time that an hsp70 response to V. 130 anguillarum has been studied in cell cultures of head kidney and liver and it appears that the SHK-1 cell line may be less sensitive in its responses compared to the primary hepatocyte culture. Long term cell lines may not reflect the original tissues as the composition of the cell population likely changes significantly over time and findings from immunological studies using such cell lines should be interpreted cautiously. 131 References Ackerman, P. A., R. B. Forsyth, C. F. Mazur and G. Iwama. 2000. Stress hormones and the cellular stress response in salmonids. Fish Physiology and Biochemistry 23: 327-336. Ackerman, P. A. and G. K. Iwama. 2001. Physiological and cellular stress responses of juvenile rainbow trout to vibriosis infection. Journal of Aquatic Animal Health 13: 173-180. Ackerman, P. A., J. C. Thornton and G. K. Iwama. 2000. Physiological and immunological effects of adjuvanted Aeromonas salmonicida vaccines on juvenile rainbow trout. Journal of Aquatic Animal Health 12:157-164. Anderson, D. P. 1992. Immunostimulants, adjuvants, and vaccine carriers in fish: applications to aquaculture. Annual Review of Fish Diseases 2: 281-307. Bachelet, M., C. Adrie and B. S. Polla. 1998. Macrophages and heat shock proteins. Research in Immunology 149: 727-732. Basu, N., T. Nakano, E. G. Grau and G. Iwama. 2001. The effects of Cortisol on heat shock protein 70 levels in two fish species. General and Comparative Endocrinology 124: 97-105. Bergmeyer, H. U. 1974. Lactate dehydrogenase assay with pyruvate and NADH. Methods in Enzymatic Analysis. H. U. Bergmeyer. New York, Academic Press. Vol 2: 574-579. Bogwald, J., R. A. Dalmo, R. McQueen Leifson, E. Stenberg and A. Gildberg. 1996. The stimulatory effect of a muscle protein hydrolysate from Atlantic cod, Gadus morhua L., on Atlantic salmon, Salmo salar L., head kidney leucocytes. Fish and Shellfish Immunology 6: 3-16. Boone, A. A. and M. M. Vijayan. 2002. Glucocorticoid mediated attenuation of the hsp-70 response in trout hepatocytes involves the proteosome. American Journal of Physiology Regulatory and Integrative Comparative Physiology 283: R680-R687. Chen, S. C , T. Yoshida, A. Adams, K. D. Thompson and R. H. Richards. 1998. Non-specific immune response of Nile tilapia, Oreochromis nilotica, to the extracellular products of Mycobacterium, spp. and to various adjuvants. Journal of Fish Diseases 21: 39-46. Ciavarra, R. and A. Simeone. 1990. T lymphocyte stress response. Cellular Immunology 129: 363-376. Currie, S., C. D. Moyes and B. L. Tufts. 2000. The effects of heat shock and acclimation temperature on hsp70 and hsp30 mRNA expression in rainbow trout : in vivo and in vitro comparisons. Journal of Fish Biology 56: 398-408. Dalmo, R. A. and J. Bogwald. 1996. Distribution of intravenously and perorally administered Aeromonas salmonicida lipopolysaccharide in Atlantic salmon, Salmo salar L. Fish and Shellfish Immunology 6: 427-441. Dalmo, R. A., K. Ingbrigsten and J. Bogwald. 1997. Non-specific defence mechanisms in fish, with particular reference to the reticuloendothelial system (RES). Journal of Fish Diseases 20: 241-273. Dalmo, R. A., T. Seternes, S. M. Arnesen, T. 6. Jorgensen and J. B0gwald. 1998. Tissue distribution and cellular uptake of Aeromonas salmonicida lipopolysaccharide (LPS) in some marine fish species. Journal of Fish Diseases 21: 321-334. 132 Dannevig, B. H., K. Falk and E. Namork. 1995. Isolation of the causal virus of infectious salmon anaemia (ISA) in a long-term cell line from Atlantic head kidney. Journal of General Virology 76: 1353-1359. Del Rio-Rodriguez, R. E., V. Inglis and S. D. Millar. 1997. Survival of Escherichia coli in the intestine off ish. Aquaculture Research 28: 257-264. Demers, N. E. and C. J. Bayne. 1997. The immediate effect of stress on hormones and plasma lysozyme in rainbow trout. Developmental and Comparative Immunology 21: 363-373. Espenes, A., M. C. Press, L. J. Reitan and T. Landsverk. 1996. The trapping of intravenously injected extracellular products from Aeromonas salmonicida in head kidney and spleen of vaccinated and nonvaccinated Atlantic salmon, Salmo salar L. Fish and Shellfish Immunology 6: 413-426. Fevolden, S. E., T. Refstie and K. H. Roed. 1992. Disease resistance in rainbow trout (Oncorhynchus mykiss) selected for stress response. Aquaculture 104: 19-29. Forsyth, R. B., E. P. M. Candido, S. L. Babich and G. K. Iwama. 1997. Stress protein expression in coho salmon with Bacterial Kidney Disease. Journal of Aquatic Animal Health 9: 18-25. Harbell, S. C , H. O. Hodgins and M. H. Schiewe. 1979. Studies on the pathogenesis of vibriosis in coho salmon Oncorhynchus kisutch (Walbaum). Journal of Fish Diseases 2: 391-404. Iwama, G. K., M. M. Vijayan, R. B. Forsyth and P. A. Ackerman. 1999. Heat shock proteins and physiological stress in fish. American Zoologist 39: 901-909. Jaquier-Sarlin, M. R., K. Fuller, A. T. Dinh-Xuan, M. J. Richard and B. S. Polla. 1994. Protective effects of hsp70 in inflammation. Experientia 50: 1031-1038. Koban, M., G. Graham and C. L. Prosser. 1987. Induction of heat shock protein synthesis in teleost hepatocytes: Effects of acclimation temperature. Physiological Zoology 60: 290-296. Lindquist, S. 1988. The heat shock proteins. Annual Review of Genetics 22: 631-677. Litwack, G. 1955. Photometric determination of lysozyme activity. Proceedings for the Society for Experimental Biology and Medicine 89: 401-403. MacArthur, J. I., A. W. Thomson and T. C. Fletcher. 1985. Aspects of leucocyte migration in the plaice Pleuronectes platessa L. Journal of Fish Biology 27: 667-676. Maule, A. G., R. Schrock, C. Slater, M. S. Fitzpatrick and C. B. Schreck. 1996. Immune and endocrine responses of adult chinook salmon during freshwater immigration and sexual maturation. Fish and Shellfish Immunology 6: 221-233. Mock, A. and G. Peters. 1990. Lysozyme activity in rainbow trout, Oncorhynchus mykiss (Walbaum), stressed by handling, transport and water pollution. Journal of Fish Biology 37: 873-885. Mommsen, T. P., T. M. Moon and P. J. Walsh. 1994. Hepatocytes: isolation, maintenance and utilization. Biochemistry and Molecular Biology of Fishes: Analytical Techniques. P. W. Hochachka and T. P. Mommsen. New York. Elsevier Science B.V. 3. Morimoto, R. I., A. Tissieres and C. Georgopoulos. 1990. The Stress Response, Function of the Proteins, and Perspectives. Stress Proteins in Biology and Medicine. R. I. Morimoto. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. 133 Nakano, K. and G. Iwama. 2002. The 70-kDa heat shock protein response in two intertidal sculpins Oligocottus maculosus and O. snyderi: relationship of hsp70 and thermal tolerance. Comparative Biochemistry and Physiology A 133: 79-94. Ritossa, F. 1962. A new puffing pattern induced by temperature shock and DNP in Drosophila. Experientia 18: 571-573. Sadovskaya, I., J. Brisson, E. Altman and L. M. Mutharia. 1996. Structural studies, of the lipopolysaccharide O-antigen and capsular polysaccharide of Vibrio anguillarum serotype 0:2. Carbohydrate Research 283: 111-127. Salati, F., M. Hamaguchi and R. Kusuda. 1987. Immune response of red sea bream to Edwardsiella tarda antigens. Fish Pathology 22: 93-98. Sanders, B. M. 1990. Stress Proteins: potential as multitiered biomarkers. Environmental Biomarkers. L. Shugart and J. McCarthy. Florida, Lewis Publishers: 165-191. Sanders, B. M. 1993. Stress proteins in aquatic organisms: an environmental perspective. Critical Reviews in Toxicology 23: 49-75. Schett, G., K. Redlich, Q. Xu, P. Bizan, M. Groger, M. Tohidast-Akrad, H. Kiener, J. Smolen and G. Steiner. 1998. Enhanced expression of heat shock protein 70 (hsp70) and heat shock factor 1 (HSF1) activation in rheumatoid arthritis synovial tissue. Journal of Clinical Investigation 102: 302-311. Selye, H. (1967). In vivo: the case for supramolecular biology. New York: Liveright. Solem, S. T., J. B. Jorgensen and B. Robertsen. 1995. Stimulation of respiratory burst and phagocytic activity in Atlantic salmon (Salmo salar, L.) macrophages by lipopolysaccharide. Fish and Shellfish Immunology 5: 475-491. Stensvag, K., J. Bogwald, B. Smedsrod and T. O. Jorgensen. 1999. Distribution of intravenously injected A-layer protein and lipopolysaccharide (LPS) from Aeromonas salmonicida in Atlantic salmon, Salmo salar L. Fish and Shellfish Immunology 9: 591-607. Stolen, J. S., T. C. Fletcher, D. P. Anderson, B. S. Roberson and W. B. van Muiswinkel, Eds. 1990. Techniques in Fish Immunology. Fish Immunology Technical Communications. Fair Haven, NJ, SOS Publications. 134 GENERAL DISCUSSION The experiments presented in this thesis were conducted with both pathogenic and non-pathogenic immune challenge, examined both cellular and physiological stress responses, and covered levels of biological organization from whole animal to cellular. From this diversity of experimental approaches, there are a number of general observations that can be made regarding the study of physiological responses to immunological stimulation in salmonids. Aquatic toxicological standards are set in the hope that sensitive organisms will be protected. Much research has been performed to determine appropriate levels, but such studies are often criticized with respect to their methodology and interpretation. In standard toxicological studies, unfed, unstressed animals are held in static water conditions and exposed to noxious elements for a period of (normally) 96 h. The ammonia exposure study (Chapter 1) demonstrated that sublethal effects may have more subtle biological consequences such as reduced resistance to infection. Levels of aquatic ammonia well below those set out in the EPA guidelines were responsible for a range of stress responses. There was an early increase in activity of neutrophils in blood (as established by NBT activity), indicating that the non-specific immune system had been mobilized, but this was transient and was not observed after a 10 d exposure. Significant alterations in Cortisol and glucose occurred at 96 h and lasted to the end of the study. Transient changes in hsp70 at 48 h were different in two tissues examined and it was found that fish exposed to ammonia had an increased mortality when subjected to a disease challenge with the pathogen V. anguillarum. Interestingly, fish exposed to the higher dose had a lower hsp70 response and also had the greatest mortality. Results suggest that there is a greater cost to ammonia exposure than has been recognized, and that the standards require further investigation to address these costs. A disease challenge (Chapter 2) was used to investigate changes that occurred in the physiology of fish during a pathogenic challenge and a number of physiological and cellular stress responses were observed. It is perhaps not surprising that V. anguillarum infection is perceived at this level as stressful, yet the responses have not been previously catalogued. The fact that hsp70 increased prior to any clinical signs of infection may be interpreted in several ways. Possibly it represents an increased production in an attempt to maintain cellular integrity during cellular damage brought about by an increase in bacterial load in the blood. However, hsp70 began to increase prior to mortalities or to peak bacterial concentrations. Another possibility is that hsp may have been somehow involved in an immune response. While it was not specifically investigated in this thesis, the increase in hsp70 could be due to 135 oxidative damage brought on by an inflammatory response. Inflammation is known to result in hsp70 accumulation (Jaquier-Sarlin et al. 1994; Polla et al. 1996; Chen et al. 1999). Exposure of fish to immune stimulation (Chapter 3) using bacterial LPS and a bacterin based on the same V. anguillarum isolate was performed in an attempt to clarify whether stress responses were limited to a live infection. LPS has been used as an immunomodulator in several fish vaccine trials (Baba ef al. 1988; Anderson 1992; Joosten ef al. 1996; Ackerman ef al. 2000) and it has not been shown to have any detrimental effects on salmonids at concentrations up to 355 mg/kg (Harbell ef al. 1979). In the current study, it was found that LPS application resulted in an early transient Cortisol increase at 24 h, but that this returned to control levels by 48 h while the bacterin had a more significant effect on plasma glucose levels. Hsp70 levels increased significantly in the head kidney in response to LPS during the first 3 d post injection while both treatment groups showed a marked decrease in the liver during this same period. Several researchers have shown a dampening effect of Cortisol on hsp production in fish (Ackerman ef al. 2000; Boone et al. 2002; Basu ef al. 2003). Cortisol is also responsible for suppression of specific immune responses during a stress event (Maule ef al. 1989) and it was thought that the observed Cortisol increase was having a similar dampening effect on hsp70 response during an infection. It was hypothesized that by removing physiological effects of Cortisol, a significant increase in hsp70 levels would be seen during an infection. The blocker RU-486 has been reported to have no effect on Cortisol production (Reddy ef al. 1995) yet in the present study it was found that in the absence of any stressor, Cortisol levels in the plasma were increased over controls and that infected fish had an even greater level of Cortisol in circulation. RU-486 is an antagonistic steroid analogue that binds to the glucocorticoid receptor (Schulz ef al. 2002) thus blocking physiological responses to Cortisol. While it did mitigate metabolic (as measured by glucose) changes in response to an infection, fish receiving the blocker had a 15% greater mortality rate than sham challenged fish. No studies have been undertaken in fish to examine immunological effects of RU-486, but mammalian studies have suggested that its use has a depressive effect on the immune system, perhaps due to an elevation of circulating Cortisol. Although physiological effects may be mitigated, Cortisol may act differentially on immunological components. When Cortisol was present at biologically natural levels (Chapter 2) hsp70 increased in both tissues. With respect to this, the results of the blocking study were unexpected. Hsp70 had a relatively similar increase in the head kidney although even the presence of a sham carrier was enough to result in a similar increase. This is perhaps not surprising as adjuvants based on oils are known to cause granulomatous lesions. However, similar to what was seen in the non-lethal challenges 136 (Chapter 3), there was a marked decrease in levels in the liver of fish that were challenged in the presence of RU-486. The experiments in Chapter 5 concentrated on the stress responses of cells, both in the presence and absence of Cortisol, from two tissues examined throughout this thesis. Again there were differences specific to the two cell types. SHK-1 cells derived from the head kidney of Atlantic salmon (Dannevig etal. 1997) showed cellular damage, as indicated by LDH actvity, was most evident 24 h after a live challenge, yet hsp70 levels were greater at 4 h. It is likely that this is due to a reduced ability of these cells to function 24 h into a challenge. It is interesting though that cells exposed to E. coli LPS had a greater hsp70 level at 4 h than 24 h and that this difference was abolished by addition of Cortisol. There was no statistical difference between 4 and 24 h levels in cells exposed to V. anguillarum LPS alone although the trend was similar. When Cortisol was combined with this treatment the difference was significant between the two times. Addition of Cortisol resulted in a similar decrease in hsp70 levels between 4 and 24 h in cells exposed to either killed bacterin or live pathogen which may indicate that a dampening effect similar to that which has been previously reported may have occurred. However, in all these treatments there was a trend towards greater LDH activity in the supernatant at 24 h suggesting a reduced hsp70 response at this time may have been due to cellular damage. Primary cultures of hepatocytes responded quite differently to challenge. Although E. coli LPS still resulted in a significant change in hsp70 levels, it did so both in the presence and absence of Cortisol, and responses were greater at 24 h than at 4 h. Similar to the SHK-1 study, all three challenges using V. anguillarum, live, killed, or LPS derived from it, in the presence of Cortisol, resulted in an increase in hsp70 at 24 h. RU-486 blocks the physiological effects of Cortisol and fish challenged with V. anguillarum in the presence of RU-486 (Chapter 4) had decreased amounts of hsp70 in liver tissue compared with other groups even though they had levels of circulating Cortisol that were in the physiological stress range of 155 - 200 ng/mL. If Cortisol does have an effect on hsp70 during an infection, it appears to be a positive relationship rather than a negative one, at least in this tissue, such that an increase in biologically available Cortisol corresponds to an increase in liver hsp70. Historically, the fields of physiology, endocrinology, and immunology have been considered quite separate. It is apparent from the studies contained within this thesis that there are important interactions that require further investigation and that it is becoming increasingly difficult to separate physiological responses from immunological ones. While physiological studies have examined the relationships between stress and disease resistance and some factors associated with it, immunological studies have rarely crossed over to examine 137 physiological responses to infection. All of the systems in an organism are linked and it is these relationships that are so fascinating to investigate. Pathogenic challenge is one of the most fundamental stressors and, as such, deserves to be studied from all angles. Hsp70 is known to increase in response to a chronic infection with Renibacterium salmoninarum but the increase was associated with regions of tissue damage in fish with clinical signs of disease (Forsyth et al. 1997). The experiments in this thesis were carried out using an acute infectious agent and have demonstrated that physiological and cellular responses are seen very early on in infection, and in individuals with no clinical signs of disease. The research contained within this thesis provides new insights into the pathogenesis of acute infectious disease and the means by which an organism reacts to early stages of infection. Comments on Future Research Aquaculture is a much maligned global industry. Current events place it in many lights, unfavourably more often than not. Fears of contaminated feeds, tainted flesh, misuse of antibiotics, and as a vessel for disease transmission are just some difficulties faced. Use of antibiotics and chemical therapeutants represent some of the most controversial of these topics due to potential effects on the environment and other species. The fact that stress increases disease susceptibility further exacerbates this issue. While chronic infections may provide a window for treatment, this is generally not the case for acute infections that have a very short time frame between onset of disease and lethal consequences. Vaccines have historically been proven to reduce incidence of disease and prevent outbreaks that are harmful not only to cultured stocks, but also to wild species in the vicinity. There are pathogens to which we have been unable to formulate vaccines due to their nature, but ongoing research suggests that some of these may be on the near horizon. A clearer understanding of physiological processes underlying these diseases could lead to investigations into use of hsps as adjuvants in vaccines against those pathogens for which it is currently difficult or impossible to immunize. A first step in this direction could be to undertake a study in which peptide-binding hsps purified from virally- or bacterially-infected fish could be tested as immunogens. Most experience with hsps, in terms of their purification and use as a tumour vaccine in amphibians, has been with gp96 (Robert et al. 1999). The protein gp96 offers a distinct advantage over hsp70 in the development of an hsp-based vaccine since gp96 is not expressed by bacteria whereas hsp70 is. Thus, one does not need to be concerned with 138 the origin of the hsp in a putative vaccine. Animals could be challenged with a live pathogen, tissues harvested and gp96 purified. This protein could then be reintroduced to fish in a saline carrier as a vaccine. If gp96 works as a vaccine it may be because it chaperones peptides and presents them to the host innate and/or adaptive immune system. Some of the most difficult vaccines to generate are those against viral pathogens. This is because the antigenic epitopes are often small, and those that are not small often mutate. Because of the unique nature of hsps, they may prove to be valuable additions to the arsenal of materials for producing protective fish vaccines in the future. 139 References Ackerman, P. A., R. B. Forsyth, C. F. Mazur and G. Iwama. 2000. Stress hormones and the cellular stress response in salmonids. Fish Physiology and Biochemistry 23: 327-336. Ackerman, P. A., J. C. Thornton and G. K. Iwama. 2000. Physiological and immunological effects of adjuvanted Aeromonas salmonicida vaccines on juvenile rainbow trout. Journal of Aquatic Animal Health 12:157-164. Anderson, D. P. 1992. Immunostimulants, adjuvants, and vaccine carriers in fish: applications to aquaculture. Annual Review of Fish Diseases 2: 281-307. Baba, T., J. Imamura, K. Izawa and K. Ikeda. 1988. Immune protection in carp, Cyprinus carpio L , after immunization with Aeromonas hydrophila crude lipopolysaccharide. Journal of Fish Diseases 11: 237-244. Basu, N., C. J. Kennedy and G. Iwama. 2003. The effects of stress on the association between hsp70 and the glucocorticoid receptor in rainbow trout. Comparative Biochemistry and Physiology A 134: 655-663. Boone, A. A. and M. M. Vijayan. 2002. Glucocorticoid mediated attenuation of the hsp-70 response in trout hepatocytes involves the proteosome. American Journal of Physiology Regulatory and Integrative Comparative Physiology 283: R680-R687. Chen, W., U. Syldath, K. Bellmann, V. Burkart and H. Kolb. 1999. Human 60-kDa heat-shock protein: a danger signal to the innate immune system. J Immunol 162: 3212-9. Dannevig, B. H., B. E. Brudeseth, T. Gjoen, M. Rode, H. I. Wergeland, O. Evensen and E. M. Press. 1997. Characterization of a long term cell line (SHK-1) developed from the head kidney of Atlantic salmon (Salmo salar L ) . Fish and Shellfish Immunology 7: 213-226. Forsyth, R. B., E. P. M. Candido, S. L. Babich and G. K. Iwama. 1997. Stress protein expression in coho salmon with Bacterial Kidney Disease. Journal of Aquatic Animal Health 9: 18-25. Harbell, S. C , H. O. Hodgins and M. H. Schiewe. 1979. Studies on the pathogenesis of vibriosis in coho salmon Oncorhynchus kisutch (Walbaum). Journal of Fish Diseases 2: 391-404. Jaquier-Sarlin, M. R., K. Fuller, A. T. Dinh-Xuan, M. J. Richard and B. S. Polla. 1994. Protective effects of hsp70 in inflammation. Experientia 50: 1031-1038. Joosten, P. H. M., W. J. Kruijerand J. H. W. M. Rombout. 1996. Anal immunisation of carp and rainbow trout with Vibrio anguillarum bacterin. Fish and Shellfish Immunology 6: 541-551. Maule, A. G., R. A. Tripp, S. L. Kaattari and C. B. Schreck. 1989. Stress alters immune function and disease resistance in chinook salmon (Oncorhynchus tshawytscha). Journal of Endocrinology 120: 135-142. Polla, B. S. and A. Cossarizza. 1996. Stress proteins in inflammation. Stress Inducible Cellular Responses. U. Feige, R.I. Morimoto, I. Yahara and B. Polla. Basel, Switzerland, Birkhauser Verlag: 375-392. Reddy, P. K., M. M. Vijayan, J. F. Leatherland and T. W. Moon. 1995. Does RU486 modify hormonal responses to handling stressor and Cortisol treatment in fed and fasted rainbow trout? Journal of Fish Biology 46: 341-359. 140 Robert, J., A. Menoret, P. Srivastava and N. Cohen. 1999. The cell surface expression of the endoplasmic reticular heat shock protein gp96 by some normal lymphoid cells is conserved during evolution. Journal of Immunology 163: 4133-4139. Schulz, M., M. Eggert, A. Baniahmad, A. Dostert, T. Heinzel and R. Renkawitz. 2002. RU486 induced glucocorticoid receptor agonism is controlled by the receptor N terminus and by coreceptor binding. Journal of Biological Chemistry 277: 26236-26243. Experience, the most brutal of teachers; but you learn, my God do you learn. — CS. Lewis 

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