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The effects of stress, cortisol, and pulp mill effluent on phagocyte function and disease resistance… Pegg, James R. 1996

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THE EFFECTS OF STRESS, CORTISOL, A N D PULP M I L L E F F L U E N T O N P H A G O C Y T E FUNCTION A N D DISEASE RESISTANCE IN J U V E N I L E SALMONIDS by JAMES R. P E G G B.Sc. University of Victoria, 1992 A THESIS IN P A R T I A L F U L F I L L M E N T OF T H E REQUIREMENTS FOR THE D E G R E E OF M A S T E R OF SCIENCE in THE F A C U L T Y OF G R A D U A T E STUDIES (Department of Animal Science) We accept this thesis as conforming to the required standard  THE UNIVERSITY OF BRITISH C O L U M B I A July 1996 © James Robert Pegg . 1996  In  presenting this  degree at the  thesis in  University of  partial  fulfilment  of  of  department  this thesis for or  by  his  or  scholarly purposes may be granted her  representatives.  permission.  Department The University of British Columbia Vancouver, Canada  for  an advanced  Library shall make it  agree that permission for extensive  It  publication of this thesis for financial gain shall not  DE-6 (2/88)  requirements  British Columbia, I agree that the  freely available for reference and study. I further copying  the  is  by the  understood  that  be allowed without  head of copying  my or  my written  Abstract  Several experiments were performed to determine the effect of confinement stress, Cortisol, and pulp mill effluent on phagocyte function in juvenile salmonids. Phagocytosis and superoxide production of anterior kidney phagocytes as well as plasma Cortisol and plasma glucose concentrations were measured. Five different confinement experiments and a disease challenge were performed, but only a combination of confinement and increased temperature resulted in a decrease in phagocytosis at 3 d. However, superoxide production was increased at the same time and at two other times in separate experiments. In a disease challenge experiment, confinement had no effect on mortality due to vibriosis, however, mortalities due to an opportunistic infection of tail rot were significantly higher in unconfined, sham-challenged fish suggesting that in this case confinement was protective. Plasma glucose concentration was also significantly elevated in unconfined, sham-challenged fish 2 d post challenge while plasma Cortisol concentration was elevated in both confined and unconfined disease-challenged fish. Phagocytosis was increased in unconfined disease-challenged fish at 2 d post challenge while superoxide production was increased in both confined and unconfined shamchallenged fish. These results indicate that the relationship between stress, immune function, and disease resistance is complex, and requires the measurement of many aspects of immune function. Injection of a Cortisol analog (prednisolone) and implantation of Cortisol into coho salmon (Oncorhynchus kisutch) resulted in decreased phagocytosis. Cortisol implantation in chinook salmon (O. tshawytscha), however, resulted in increased phagocytosis and had no effect on superoxide production. Cortisol had no effect on phagocytosis or superoxide production in vitro at a physiological concentration, however, phagocytes incubated in vitro with 10% pooled serum from fish stressed for 1 h or 3 d resulted in a significant reduction in phagocytosis and an increase in superoxide production compared to  ii  phagocytes incubated with serum from control fish. This combined with the Cortisol results from the confinement experiments indicate that the effect of Cortisol is not direct and that plasma Cortisol concentration is not a good predictor of phagocyte function. Pulp mill effluent caused significant increases in superoxide production and decreases in phagocytosis especially at low concentrations. Hypoxia also caused an increase in superoxide production, but had no effect on phagocytosis. It was concluded that stress and Cortisol do not have a general suppressive effect on immune function. Some aspects of immune function may be suppressed while others are stimulated to compensate. Pulp mill effluent exposure had a significant effect on phagocyte function, and like stress and Cortisol, both suppressive and stimulatory effects were observed.  iii  Table of Contents Abstract  ii  Table of Contents  iii  List of Tables  iv  List of Figures  v  Acknowledgments  vii  General Introduction  1  General Methods  8  Chapter 1 Effects of Stress on Phagocyte Function and Disease Resistance in Juvenile Chinook Salmon (Oncorhynchus tshawytscha)  12  Introduction  13  Materials and Methods  15  Results  19  Discussion  29  Chapter 2 Effects Cortisol on Phagocyte Function in Juvenile Chinook (Oncorhynchus tshawytscha) and Coho Salmon(0. kisutch)  35  Introduction  36  Materials and Methods  38  Results  42  Discussion  49  Chapter 3 The Effects of a 30 Day Exposure to Pulp M i l l Effluent on Phagocyte Function in Juvenile Chinook Salmon (Oncorhynchus tshawytscha)  53  Introduction  54  Materials and Methods  57  Results  60  Discussion  65  Concluding Remarks  67  References  71  iv  List of Tables  Table 1. Experimental conditions in the confinement experiments  17  Table 2. Plasma lysozyme concentrations of juvenile chinook salmon, Oncorhynchus tshawytscha, confined in 10 L of water at room temperature with aeration  20  Table 3. Experimental conditions of Cortisol experiments  v  40  List of Figures Figure 1-1. Diagram of treatments used in the confinement experiments: confinement and increased temperature (A), low density confinement (B), high density confinement (C), medium density confinement (D)  16  Figure 1-2. The effects of confinement and temperature on plasma Cortisol concentration (A), plasma glucose concentration (B), phagocytosis (C), and superoxide production (D) of adherent phagocytes from the anterior kidney of juvenile chinook salmon, Oncorhynchus tshawytscha  22  Figure 1-3. The effects of confinement on plasma Cortisol concentration (A), plasma glucose concentration (B), phagocytosis (C), and superoxide production (D) of adherent phagocytes from the anterior kidney of juvenile chinook salmon, Oncorhynchus tshawytscha  23  Figure 1-4. The effects of confinement stress on plasma Cortisol concentration (A), plasma glucose concentration (B), phagocytosis (C), and superoxide production (D) of adherent phagocytes from the anterior kidney of juvenile chinook salmon, Oncorhynchus tshawytscha  24  Figure 1-5. The effects of confinement stress on plasma Cortisol concentration (A), plasma glucose concentration (B), phagocytosis (C), and superoxide production (D) of adherent phagocytes from the anterior kidney of juvenile chinook salmon, Oncorhynchus tshawytscha  25  Figure 1-6. The effects of confinement stress on plasma Cortisol concentration (A), plasma glucose concentration (B), phagocytosis (C), and superoxide production (D) of adherent phagocytes from the anterior kidney of juvenile chinook salmon, Oncorhynchus tshawytscha  26  Figure 1-7. The effects confinement stress on mortality of chinook salmon, Oncorhynchus tshawytscha, following an immersion challenge with Vibrio anguillarum in aerated peptone saline. Disease mortalities were confirmed as vibriosis by culture 27 Figure 1-8. The effects confinement stress and disease challenge on plasma Cortisol concentration (A), plasma glucose concentration (B), phagocytosis (C), and superoxide production (D) of adherent phagocytes from the anterior kidney of juvenile chinook salmon, Oncorhynchus tshawytscha  vi  28  Figure 2-1. The effects of a single Cortisol injection (prednisolone 20mg/g) in the dorsal sinus of juvenile coho salmon, Oncorhynchus kisutch, on percent phagocytosis of glass adherent phagocytes isolated from the anterior kidney over time  44  Figure 2-2. The effects of Cortisol implantation (100 p.g/g) on percent phagocytosis in juvenile coho salmon  45  Figure 2-3. The effects of 2 doses of Cortisol implants (50 and 100 ug/g) and sham implants on plasma Cortisol concentrationA), plasma glucose concentration(B), phagocytosis(C), and superoxide production (D) of adherent anterior kidney phagocytes isolated from juvenile chinook salmon, Oncorhynchus tshawytscha  _7 Figure 2-4. In vitro effects of Cortisol (181 ng/ml / 5x10 M ) on phagocytosis and superoxide production in phagocytes isolated from juvenile chinook salmon  46  47  Figure 2-5. In vitro effects of 10% serum from juvenile chinook stressed for 1 h or 3 d on phagocytosis and superoxide production of phagocytes isolated from juvenile chinook salmon 48 Figure 3-1. Effects of several concentrations of pulp mill effluent and hypoxia on superoxide production (A) and phagocytosis (B) of anterior kidney phagocytes from juvenile chinook salmon (Oncorhynchus tshawytscha) Figure 3-2. Effects of several concentrations of pulp mill effluent on superoxide production (A) and phagocytosis (B) of anterior kidney phagocytes from juvenile chinook salmon (Oncorhynchus tshawytscha) under normoxic conditions Figure 3-3. Effects of several concentrations of pulp mill effluent on plasma Cortisol and glucose concentrations of juvenile chinook salmon (Oncorhynchus tshawytscha) under normoxic conditions  vii  62  63  64  Acknowledgments This thesis would not have been possible without the help and support of a large number of people. I would like to acknowledge my supervisor George Iwama for giving me the opportunity to work in his lab, Bob Gordon and Dennis Martens ,who got me started in this field, for all of their support and encouagement, George Kruzynski for inviting me to sample fish from his pulp mill experiment which he has spent many long, cold months setting up in Prince George, my comittee members, Jim Thompson and Alec Maule, for reviewing my thesis, Dave Shackleton for ensuring that my defense happened when it was supposed to, my parents for their encouragement, all the fish who gave up their lives in the name of science, and everyone in the fishlab for all of their help and support. A portion of this data was presented at the Modulators of Immune Responses conference in Breckenridge Colorado in July 1995, at the Aquaculture Association of Canada meeting in Nanaimo in June 1995, and at the International Congress on the Biology of Fishes in San Francisco in July 1996, and has been published in proceedings papers (Pegg et al, 1995; Pegg and Iwama, 1996). Financial support for this work was provided by a scholarship from N S E R C to J.R.P. and an operating grant from the Canadian Bacterial Diseases Network to G.K.I. Additional support was received from the Department of Fisheries and Oceans, Cultus Lake Research Lab.  vin  General Introduction  Salmonids are an important resource in British Columbia for commercial fisheries, aquaculture, sports fishing, and tourism. These fish encounter  stressful  situations frequently both in the wild and in aquaculture (Pickering 1989). Therefore, understanding the effects of stressors on fish will provide valuable information for managing fish, both in aquaculture and in the wild. Stress has been defined in a variety of ways (see Barton and Iwama 1991; Adams 1990). The definition proposed by Brett (1958) states that "Stress may be defined as a state produced by an environmental or other factor which extends the adaptive responses of an animal beyond the normal range or which disturbs the normal functioning to such an extent that, in either case, the chances of survival are significantly reduced". It may also be argued that "stress" does occur before the chances of survival are significantly reduced. A stressor is the stimulus that causes stress, and the stress response is the sum of the changes that occur in response to a stressor. The stress response can be broadly categorized into primary, secondary and tertiary  responses  (Barton and  Iwama  1991).  Primary responses include  the  neuroendocrine / endocrine responses including alterations in blood catecholamines, adrenocorticotropic hormone (ACTH), and Cortisol concentrations. The secondary effects are mediated partly as a result of the primary responses and include alterations in blood glucose levels, hematology, hydromineral balance, immune function, and metabolism. Tertiary effects include the whole animal effects such as changes in growth, disease  1  resistance, and reproductive potential. (Barton and Iwama 1991). Not all stress responses, however, follow this pattern since some stressors such as toxicants may not result in a Cortisol increase (Barton and Iwama 1991). A typical stress response in salmonids consists of an immediate production of catecholamines and A C T H . A C T H from the pituitary gland stimulates the interrenal cells to produce Cortisol which is regulated through the hypothalamic-pituitary axis. Plasma Cortisol levels usually peak within 1-2 h and return to baseline levels within 1-2 d depending on the species and experimental conditions (Gamperl et al. 1994). Increased plasma glucose levels are a secondary response and usually peak 3-6 h after a stressor and return to baseline levels within 1-2 d depending on the stressor. (Pickering et al. 1989). Effects of stress on immune function have been reported within 3 h (Narnaware et al. 1994) and may last several days or weeks (Maule et al. 1989). Stress in aquaculture can result from crowding or handling, but also from poor water quality and social interactions. Potential stressors in the wild include contaminants, poor water quality, changes in temperature, migration, and social interactions. The effects of stress on the physiology of fish are well documented (Barton and Iwama 1991). Although the stress response is adaptive for short term stressors encountered in the wild there are detrimental effects of severe or chronic stress which can occur in aquaculture. Of these, the decrease in immune function and disease resistance is one of the most serious. Several studies have demonstrated that stress can cause changes in immune function in fish (Thompson et al. 1993; Maule and Schreck 1990; Maule et al. 1989; Mazur and Iwama 1993; Angelidis et al. 1987; Peters et al. 1991; Peters and Schwarzer  2  1985; Narnaware et al. 1994; Ellsaessor and Glem 1986), and decreased disease resistance (Maule et al. 1989; Wise et al. 1993; Wedemeyer et al. 1984). Many authors have reported changes in immune function, mainly suppression, due to Cortisol (Pickering 1984; Anderson et al. 1982; Slater and Schreck 1993; Tripp et al. 1987; Bennet and Wolke 1987b; Ellsaesser and Clem 1987; Grim et al. 1985; Pulsford 1995; Stave and Roberson 1985). Cortisol has also been shown to decrease disease resistance in several species offish (Houghton and Matthews 1986; Maule et al. 1987; Pickering 1989; Woo etal. 1987). From such data, the conclusion is often drawn that immunosuppression and decreased disease resistance is a general consequence of stress and elevated plasma Cortisol concentrations (Schreck et al. 1993). This may be an oversimplification as it is unlikely that all stressors and elevations in plasma Cortisol concentration result in fish becoming more susceptible to disease. Assays of immune function are becoming popular tools for studying the effects of contaminants on fish since changes in immune function can affect disease resistance which can directly affect the survival of the fish. Several researchers have shown effects of environmental contaminants on various aspects of immune function (Weeks and Warinner 1984, 1986; Weeks et al. 1986; Walczak et al. 1987; Rice and Schlenk 1995; Seeley and Weeks-Perkins 1991; Anderson et al. 1989; Rice and Weeks 1989; Voccia et al. 1994a, 1994b). There are many techniques available for studying the immune system in fish (Weeks et al. 1992; Anderson 1990). Assays of non-specific immune function are one of the most relevant to fish health, because non-specific immunity represents one of the first  3  lines of defense against invading microorganisms. In addition non-specific immunity is thought to be more important than specific immunity at low temperatures and during the early life stages of fish (O'Neil 1985; Chen and Ainsworth 1991; Ainsworth et al. 1991a). Phagocytes play a complex role in the immune system, they include monocytes, macrophages, and neutrophils, and are involved in the non-specific immune system through phagocytosis (uptake), killing of microorganisms (superoxide production) and lysozyme production (a bacteriolytic enzyme). Phagocytes also play a role in the specific immune system through antigen presentation and cytokine production. Factors affecting phagocyte function can therefore have a serious impact on immune competence and disease resistance. There are a variety of assays available for measuring different aspects of phagocyte function (Blazer 1991; Anderson 1990). These include: chemotaxis (directed movement of phagocytes toward a site of infection), phagocytosis (engulfment of particles), pinocytosis (uptake of liquid droplets), superoxide production (microbial killing), hydrogen peroxide production (microbial killing), chemiluminescence (microbial killing), and bactericidal activity. Some examples of methods used to measure phagocytosis  include:  spectrophotometry,  fluorescent  and  light  microscopic counting  techniques,  fluorescent spectrophotometry, microtitre techniques, and flow  cytometry. The main differences between these techniques are the target particle used (bacteria, yeast, latex beads, etc.) and the method of quantification (microscopic counting, spectrophotometer, fluorescent spectrophotometer, microplate reader, flow cytometer). In addition the cells can be collected and isolated by a variety of methods including:  4  peritoneal elicitation and lavage, continuous or discontinuous density  gradient  centrifugation of blood, anterior kidney or spleen cells, differential adherence, etc. Other variations include: whether the cells are adherent or in suspension, type of media, temperature, serum concentration, incubation time, target to cell ratio, etc. The type of assay chosen depends on experimental objectives as well as available equipment and expertise. Slide assays are relatively simple to perform and require a minimum of specialized equipment, but the microscopic counting requires expertise, is somewhat subjective, and is very time consuming. Assays that use instruments to measure the number of particles phagocytized eliminate the subjectivity of microscopic counting and decrease the time required to process samples. These instruments are often expensive and are usually not portable which may limit their use in field situations. In addition these instruments require accurate quantification of the number of phagocytes present: There has been very little comparison between the different types of assays. New methods are usually compared to one variation of microscopic counting techniques, but few of the microscopic, photometric or fluorescent techniques have been compared to each other. A considerable amount of time was spent adapting assays of phagocyte function for use with juvenile salmonids and adapting the photometric assay of phagocytosis for use with a microplate reader. In this study both slide and microplate assays of phagocytosis and superoxide production were used. Slide assays were used in field situations and with small fish which did not yield enough cells for microplate assays. However, microplate assays were preferred since the subjectivity and considerable  5  amount of time required to read the slides were eliminated. Using microplate assays, phagocytosis, superoxide production, and quantification of adherent cells by protein content could be measured in replicate wells in the same microplate and the results read automatically in a microplate reader. Short adherence and incubation times, and low serum concentrations were chosen so that phagocytes would most likely represent their state in the fish. The anterior kidney was chosen as the source of cells due to its larger size and abundance of cells. The combination of discontinuous percoll gradients and adherence was chosen since this method made it possible to process more samples at once. Chinook salmon (Oncorhynchus tshawytscha) were chosen as the fish used in the majority of the experiments due to their tendency toward high Cortisol responses (Barton and Iwama 1991) and their commercial importance.  Hypothesis and Experimental Objectives: The hypothesis that I set out to test was that there is a period following a stressor or contaminant exposure during which phagocyte function is impaired and disease resistance is reduced and that this process is mediated by the stress hormone Cortisol. The experimental objectives of this thesis were: 1) to determine the timing and duration of the effects of confinement stress on phagocyte function; 2) to describe the role of Cortisol in modulating phagocyte function; and 3) to evaluate the potential of phagocyte function as an indicator of environmental contamination for salmonids.  6  Organization of this Thesis: This thesis consists of a series of experiments which examine the effects of stress, the stress hormone, Cortisol, and pulp mill effluent on non-specific immune function and disease resistance. In the first chapter, the effects of confinement stress on two aspects of phagocyte function, phagocytosis  and superoxide production, by anterior kidney  phagocytes, were examined in chinook salmon  and compared to changes in plasma  Cortisol and glucose concentrations. In the second chapter, the effects of the stress hormone Cortisol were studied using Cortisol injection to simulate acute stress in coho salmon (O. kisutch). Cortisol implantation was used to simulate chronic stress in coho and chinook salmon. The effects of Cortisol on phagocyte function were also studied in vitro using a physiological level of Cortisol or serum from stressed or control fish. In the third chapter, the effects of a 30 day in situ exposure to pulp mill effluent on phagocyte function were investigated.  7  General Methods  Many of the methods such as phagocyte isolation are used in two or three of the following chapters and are therfore presented here in more detail to avoid repetition. Phagocyte Isolation: Fish were killed with 200 mg/L tricaine methanesulfonate (MS222). Phagocytes were isolated according to the method of Secombes (1990). Anterior kidneys were aseptically removed and placed in Leibovitz medium (L-15) supplemented  with  10 units/mL  heparin  and 100 units  penicillin-100 mg  streptomycin/mL on ice. A cell suspension was created by gently teasing the tissue through nylon mesh (50-100 um) or by gently drawing the tissue in and out o f a l c c plastic syringe (Cortisol injection experiment). The cell suspension was enriched for phagocytes by centrifuging on a 34/51% discontinuous percoll gradient for 20 min at 400 x G at 2-4°C. The interface layer was collected and washed once in L-15 then resuspended in L-15 containing 0.1% fetal calf serum (FCS). Viable cell concentrations were determined using trypan blue exclusion and a hemocytometer. Cells were added to microplates at 10^ cells per well, or chamber slides at 5x10^ cells per well. The cells were further enriched for phagocytes by allowing them to adhere for 30 min before performing functional assays. Microplate Assays of Phagocyte Function: Microplates were divided into three sections for the measurement of phagocytosis, superoxide production, and quantification of adherent cells using a protein assay. A phagocytosis assay was performed in which the medium was replaced with L-15 + 0.1% FCS containing 2x10 Congo red stained yeast /  8  mL in a microplate adaptation of a method described by Seeley et al. (1990). The phagocytes were incubated with the yeast suspension for 2 h  after which time the  unphagocytized yeast cells were washed away with phosphate buffered saline and the adherent phagocytes were solubilized overnight at 37°C in 1.5 g/L trypsin / 0.4g/L ethylenediaminetetraacetic acid (EDTA). The red stained yeast cells released from the phagocytes were quantified at 525 nm in a microplate reader against trypsin blanks and standard solutions. Superoxide production was measured by nitro blue tetrazolium (NBT) reduction with or without phorbol myristate acetate (PMA) stimulation according to the method of Secombes (1990). The phagocytes were incubated in 1 mg/mL N B T with or without 1 ug/mL P M A in 50% L-15, 50% 0.85% saline for 2 h and then fixed in methanol, washed several times in 70% methanol, air dried, and dissolved in 100 uL 2 M K O H and 100 uL dimethylsulfoxide (DMSO). The resulting turquoise/blue colour was measured with K O H / D M S O blanks and N B T standards (Rook et al. 1985) at 620 nm in a microplate reader. Adherent cell protein was determined using a bicinchoninic acid protein assay (Smith et al. 1985). The results of the phagocytosis and N B T assays were corrected for the number of phagocytes present by dividing by the amount of protein so that results are expressed per ug protein. A l l three assays were performed in duplicate or triplicate wells for each fish sampled. Slide assays of phagocyte function: 50 uL of cell suspension was added to each well of 4 well D F A T microscope slides (Cel-line Associates Inc.) or 1 m L of cell suspension to each well of 4 well chamber slides (Lab Tek) and allowed to adhere for 30 min at 15°C in a humid chamber. Non-adherent cells were washed away with a gentle  9  stream of phosphate buffered saline (PBS) and 100 mL of 2x10° washed, autoclaved yeast/mL (phagocytosis assay) or 100 mL of 2 mg/mL N B T in 0.85% saline (superoxide assay) was added to each well. The slides were incubated for another 2 h at 15°C in a humid chamber. The phagocytosis slides were rinsed with a gentle stream of PBS to remove unphagocytized yeast and then air dried and stained with Diff Quik or giemsa. The N B T slides were examined under a microscope immediately to determine the percentage of cells reducing the N B T creating a blue colour (NBT positive). The phagocytosis slides were examined under a compound microscope and the percentage of cells containing yeast was counted. . Blood sampling: Fish were killed with an overdose of MS222 and blood was taken from the caudal vessels immediately using a heparinized syringe or hematocrit tube. Blood samples were centrifuged to separate the plasma which was stored at -50°C. Plasma Cortisol concentration was determined with a radioimmune assay (coat-a-count, Diagnostic Products Corporation), and plasma glucose concentration was determined with a microplate assay using the Trinder method (Sigma). Lysozyme was measured using the lysoplate method (Osserman and Lawlor 1966, modification by Lie et al. 1986)  Statistical analyses: For the confinement stress experiments Student's t-tests were used to compare means for each day. Two-way analysis of variance was not used for the confinement stress experiments due to the large amount of variation between sampling days. This variation was likely due to small differences in the cell isolation procedure or assays rather than actual differences in the fish so experimental fish were  10  only compared to control fish which had been processed at the same time. In cases where data was not normally distributed or variances were not equal a Mann-Whitney U test was used. Two way analysis of variance was used for the Cortisol implantation and in vitro Cortisol experiments and on arcsin square root transformed proportion data for the Cortisol injection experiment. One way A N O V A was used to compare treatments in the in vitro serum experiments and in the disease challenge experiment. Student-Newman-Keuls tests were used to determine group differences. Mortality data were compared using a Chi-square analysis. Significance level for all experiments was p<0.05 and all data are presented as means +/- standard errors. A l l statistical analysis was done using SigmaStat statistical software (Jandel Scientific, San Rafael, California, U S A ) . Due to the considerable amount of time required to isolate phagocytes and perform functional assays the sample size was usually limited to a manageable size of four fish per treatment. This small sample size made it difficult to show statistical differences and in a few cases statistically insignificant differences are discussed where there may be biological differences.  11  Chapter 1 Effects of Stress on Phagocyte Function and Disease Resistance in Juvenile Chinook Salmon (Oncorhynchus  12  tshawytscha)  Introduction  Stress is experienced frequently by fish in aquaculture (e.g. handling, crowding). Several studies have demonstrated that stress can cause changes in immune function (Thompson et al. 1993; Angelidis et al. 1987; Maule and Schreck 1990; Maule et al. 1989; Narnaware et al. 1994; Ellsaessor and Clem 1986; Mazur and Iwama 1993). These include changes in the hematopoietic tissue (Peters and Schwarzer 1985), changes in specific immune function such as a reduction in the number of antibody producing cells (Maule et al. 1989; Mazur and Iwama 1993), decreased antibody production (Thompson et al. 1993) as well as changes in nonspecific immunity such as decreased phagocytosis (Narnaware et al. 1994), decreased chemiluminescent response (Angelidis et al. 1987), decreased natural cytotoxic cell activity (Ghomeum et al. 1988), decreased leukocyte bactericidal activity and increased serum bactericidal activity (Thompson et al. 1993). Decreased disease resistance as a result of stress has also been reported for several "species of fish including chinook salmon (Maule et al. 1989; Wise et al. 1993; Sniezko 1974; Wedemeyer et al. 1984). Information on the timing and duration of changes in immune function and disease resistance as a result of stress could be used to help prevent disease outbreaks in aquaculture. Stressors in aquaculture include: handling, confinement, crowding, transport, and changes in water quality. Of these, confinement or crowding is one of the more severe and is easy to replicate in the lab. In addition it may represent a situation with which the fish are not adapted to deal. Chronic stress in the wild is most likely rare since the fish  13  will attempt to avoid stressful conditions in most cases. Fish may be confined during grading, vaccination, tagging, and transport, and may be severely crowded in some aquaculture situations. In order to determine whether confinement stress affects phagocyte function a series of experiments in which fish were stressed by confinement for various lengths of time was performed. Phagocyte function was measured using either slide or microplate assays of phagocytosis and superoxide production. Plasma glucose and Cortisol concentrations were also measured. To determine whether changes in immune function due to stress affect disease resistance, a disease challenge was performed in which sham and Vibrio anguillarum-challenged fish were either stressed by confinement or returned to their tanks after being challenged.  14  M a t e r i a l s and M e t h o d s  F i s h : Juvenile chinook salmon (Oncorhynchus tshawytscha) (average 7-59 g) were obtained from commercial hatcheries. Fish were maintained in dechlorinated Vancouver city water in 70 or 170 L tanks and fed a commercial salmon diet. The fish were acclimated for at least two weeks prior to each experiment. (Table 1). Confinement stress: In order to determine whether stress affects phagocyte function, four different stressors were used in five confinement experiments with juvenile chinook salmon: experiment 1) confinement and temperature; five fish were held in 10 L of water in a white 25 L bucket at room temperature (21°C) with aeration; experiment 2) low density confinement; four fish were mildly stressed by transferring them to a white 25 L bucket with holes floating in a tank; experiment 3) high density confinement; four fish were transferrred to the same white 25 L bucket with holes was used except that it was suspended so that there was only 5 cm of water in the bottom; experiment 4 and 5) medium density confinement; in the fourth and fifth experiments fish were transferred to smaller white 4 L buckets with holes and suspended with 5 cm of water in them. Phagocytosis,  superoxide  production, plasma  Cortisol,  glucose,  and lysozyme  (confinement and increased temperature experiment only) concentration were measured. For experimental conditions of the confinement experiments see Table 1. Slide assays of phagocytosis were used in the first medium density confinement experiment due to the small size of the fish. The microplate assay of superoxide production was improved in the high density confinement  15  Figure 1-1. Diagram of treatments used in the confinement experiments: confinement and increased  temperature (A), low density  confinement  confinement (C), medium density confinement (D).  16  (B), high  density  experiment and i n the second medium density confinement experiment to include phorbol myristate acetate ( P M A ) stimulation. Phorbol myristate acetate stimulation was not used in experiments 1 and 2 or with slide assays.  Table 1. Experimental conditions of the confinement stress experiments. Experiment # 1 is the confinement and increased temperature experiment, # 2 is the low density confinement experiment, # 3 is the high density confinement experiment, and # 4 and 5 are the medium density confinement experiments.  Exp.  Month  Stressor  # 1  March  fish Fish  Tank size  Water  Ave.  Temp.  weight  Density  28 g  37 g / L  170 L  Temperature + 6°C (bucket Confinement  21°C)  2  May  Confinement  12°C  21 g  6 g/L  170 L  3  September  Confinement  14°C  59 g  85 g / L  170 L  4  January  Confinement  6°C  7g  21 g / L  70 L  5  March  Confinement  5°C  10g  30 g / L  70 L  Disease challenge experiment: Juvenile chinook salmon were acclimated to 70 L tanks and challenged by immersion with either 1.27x10  Vibrio anguillarum for 20 m i n at  colony forming units/mL i n aerated peptone saline or peptone saline only  (sham). After the challenge, fish were either returned to their tanks or to buckets suspended i n their tanks. The confinement set-up was the same as i n the l o w density confinement experiment, except the buckets were suspended i n smaller tanks and the fish density was higher. Each treatment was replicated for a total o f eight tanks. There were  17  28 fish per tank at the start of the experiment. The average weight of the fish was 50 g, resulting in densities of 20 g/L in the unstressed fish or 78 g/L in the stressed fish at the start of the disease challenge. Four fish per tank were sampled 2 d pre-challenge and 2 d post-challenge to measure phagocyte function, plasma glucose and plasma Cortisol concentrations. The average temperature during the experiment was 11°C. Statistical analyses: For the confinement stress experiments Student's t-tests were used to compare means for each day. Two-way analysis of variance was not used for the confinement stress experiments due to the large amount of variation between sampling days. This variation was likely due to small differences in the cell isolation procedure or assays rather than actual differences in the fish so experimental fish were only compared to control fish which had been processed at the same time. In cases where data were not normally distributed or variances were not equal a Mann-Whitney U test was used. One way A N O V A was used to compare treatments in the disease challenge experiment. Student-Newman-Keuls tests were used to determine group differences. Mortality data were compared using a Chi-square analysis. Significance level for all experiments was p<0.05 and all data is presented as means +/- standard errors. A l l statistical analyses were done using SigmaStat statistical software (Jandel Scientific, San Rafael, California, USA).  18  Results  Plasma Cortisol concentration was elevated for at least the first sampling point in each confinement experiment (Figures 1-2A, 1-3A, 1-4A, 1-5A, 1-6A). A t 1 d, a sampling point common to all five experiments, plasma Cortisol concentration was roughly proportional to fish density with the highest mean Cortisol concentration in the high density confinement experiment (Figure 1-4A), and the lowest mean Cortisol concentration in the low density confinement experiment (Figure 1-3A). In the confinement and increased temperature experiment plasma Cortisol concentration was elevated in confined fish at all sampling times from 2 h to 5 d (Figure 1-2A) while in the low density confinement experiment plasma Cortisol concentration was only elevated at 2 h and had returned to control levels by 1 d (Figure 1-3A). In the high and medium density confinement experiments plasma Cortisol concentration was elevated for up to 1 d and had returned to control levels by 2 d. The highest plasma Cortisol concentrations were measured in the confinement and increased temperature experiment at 2 h (240 ng/mL) (Figure 1-2 A). No difference in plasma lysozyme concentration was observed between stressed and unstressed fish (Table 1-2). Plasma lysozyme concentration was not measured in the other experiments due to the small volumes of plasma collected.  19  Table 2. The effects of confinement (37 g/L) and increased temperature (21°C) on plasma lysozyme concentrations (units/mL) in juvenile chinook salmon, Oncorhynchus tshawytscha. Means (+/- 1 S.E.), N=4. Factor  2h  3d  control  162.0 ±23.2  202.3 ± 78.8  stress  106.4 ± 12.5  184.8 ± 4 0 . 9  Plasma glucose concentration was elevated in confined fish in at least one sampling point for each confinement experiment. In the confinement and increased temperature experiment plasma glucose concentration was elevated in confined fish at 2 h and in control fish at 5 d, but not 1 d and 3 d (Figure 1-2B). In the low density confinement experiment plasma glucose concentration was elevated in confined fish at 5 d, but not at 2 h or 1 d (Figure 1-3B). In the high and medium density confinement experiments plasma glucose concentration was elevated in confined fish at 1 d and 2 d (Figure 1-4B, 1-5B, 1-6B). In the second medium density confinement experiment plasma glucose concentration was elevated by 3 h but was at control levels again at 6 h and again elevated in confined fish at 1 d and 2 d (Figure 1-6B). Phagocytosis was only significantly different in confined fish in the confinement and increased temperature experiment at 3 d where it was lower (Figure 1-2C). A t the same time superoxide production was increased (Figure 1-2D). Superoxide production was also increased at 2 d in the high density confinement experiment (Figure 1-4D) which had the highest density, and at 2 h in the second medium density confinement  20  experiment (Figure 1-6D). Increased superoxide production was also observed in the first medium density confinement experiment (Figure 1-5D) at 5 d using a slide assay. No differences in phagocytosis or superoxide production were observed in the low density confinement experiment (Figure 1-3C, 1-3D). There were no pre-challenge/pre-stress tank differences in phagocyte function or plasma glucose and Cortisol concentrations. Confinement stress had no effect on mortality due to vibriosis (Figure 1-7). Mortality due to vibriosis started 3 d post-challenge and stopped 8 d post-challenge with the majority of the mortalities occurring before 5 d postchallenge. Mortalities due to an opportunistic infection, tail rot, started 10 d postchallenge and were significantly higher in the unconfined sham-challenged fish than in the confined sham-challenged fish or in the disease challenged fish (Figure 1-7). These mortalities were characterized by severe erosion of the caudal peduncle and lack of Vibrio anguillarum isolated from the kidney. Plasma Cortisol concentrations were significantly higher in the disease-challenged fish 2 d post-challenge compared to the sham-challenged fish and pre-challenge fish (Figure 1-8A). Plasma glucose concentrations were only significantly higher in the unstressed sham-challenged fish (Figure 1-8B). Phagocytosis was significantly higher in the unstressed disease-challenged fish compared to the stressed disease-challenged fish as well as the sham-challenged fish and pre-challenge samples (Figure 1-8C). Superoxide production was significantly higher in the shamchallenged fish compared to the disease-challenged fish and the pre-challenged sample (Figure 1-8D). The stressed and unstressed sham-challenged fish were not significantly different from each other.  21  Figure 1-2. The effects of confinement and increased temperature (37 g/L, 21°C) on plasma Cortisol concentration (A), plasma glucose concentration (B), phagocytosis (C), and superoxide production (D) of adherent phagocytes from the anterior kidney of juvenile chinook salmon, Oncorhynchus tshawytscha. Means (+/- 1 S.E.), N=4, * p<0.05.  22  Figure 1-3. The effects of low density confinement (6 g/L) on plasma Cortisol concentration (A), plasma glucose concentration (B), phagocytosis (C), and superoxide production (D) of adherent phagocytes from the anterior kidney of juvenile chinook salmon, Oncorhynchus tshawytscha. Means (+/- 1 S.E.), N=4, * p<0.05.  23  1  2  3  4  5  6  7  1  Time (days)  2  3  4  5  6  7  Time (days) #  control  — A - stress  Figure 1-4. The effects of high density confinement (85 g/L) on plasma Cortisol concentration (A), plasma glucose concentration (B), phagocytosis (C), and superoxide production (D) of adherent phagocytes from the anterior kidney of juvenile chinook salmon, Oncorhynchus tshawytscha. Means (+/- 1 S.E.), N=4, * p<0.05. 24  i  1  1  2  1  3  1  4  1  1  5  1  1  Time (days)  i  2  3  i  r  4  5  Time (days)  • — A -  control stress  Figure 1-5. The effects of medium density confinement (21 g/L) on plasma Cortisol concentration (A), plasma glucose concentration (B), phagocytosis (C), and superoxide production (D) of adherent phagocytes from the anterior kidney of juvenile chinook salmon, Oncorhynchus tshawytscha. Means (+/- 1 S.E.), N=4, * p<0.05.  25  0  10  20  30  40  50  0  Time (hours)  10  20  30  40  50  Time (hours)  •  control  — A - stress  Figure 1-6. The effects of medium density confinement (30 g/L) on plasma Cortisol concentration (A), plasma glucose concentration (B), phagocytosis (C), and superoxide production (D) of adherent phagocytes from the anterior kidney of juvenile chinook salmon, Oncorhynchus tshawytscha. Means (+/- 1 S.E.), N=4, * p<0.05.  26  t- control disease ^- stress disease stress sham — ) control sham  Figure 1-7. The effects confinement stress on mortality in juvenile chinook salmon, Oncorhynchus tshawytscha, following an immersion challenge with Vibrio anguillarum in aerated peptone saline. Mortalities were confirmed as vibriosis by culture in the disease challenged groups while mortalities in the sham-challenged groups were due to tail rot. Means, N=2, * different from sham challenged control fish at p<0.05. 27  h  6 c '53  5o i_  Q.  A O) L  ^  ->  O  1  r X  h 1  B  J_  _I_  h 0.1  pre  T  T  T  s-c  s-s  d-c  T  d-s  pre  T  s-c  s-s  d-c  d-s  Treatment  Treatment  Figure 1-8. The effects confinement stress and disease challenge on plasma Cortisol concentration (A), plasma glucose concentration (B), phagocytosis (C), and superoxide production (D) of adherent phagocytes from the anterior kidney of juvenile chinook salmon, Oncorhynchus tshawytscha. pre = pre-challenge sample, s-c = sham-challenged control, s-s = sham-challenged stressed, d-c = disease-challenged control, d-s = disease-challenged stressed. Means (+/- 1 S.E.), N=8, * p<0.05.  28  Discussion Elevated plasma Cortisol concentrations were observed for at least the first sampling point in all five confinement stress experiments, indicating the treatment was stressful. Plasma Cortisol concentration at 1 d was related to fish density with the highest concentrations observed in the highest densities. In the confinement and increased temperature experiment plasma Cortisol concentration was elevated at all sampling times indicating that this combination of confinement and increased temperature resulted in chronic stress. Plasma Cortisol concentrations in the other experiments had returned to control levels by 2 d suggesting that the stress may have been mostly due to the handling or that they may have acclimated to the new conditions. In the low density confinement experiment Cortisol levels had returned to control levels by 1 d indicating that this treatment was less stressful than the others. The chronically elevated plasma Cortisol concentrations in the confinement and increased temperature experiment are likely due to the increased temperature since in the high density confinement experiment in which the fish density was more than twice as high, plasma Cortisol concentrations had returned to control levels by 2 d. Plasma glucose concentrations were also elevated in stressed fish during at least one sampling point in each experiment, however, no clear pattern emerged. Plasma glucose levels may be more dependent on clearance so that in the confinement and increased temperature experiment which was clearly stressful from the Cortisol data, glucose may be used as quickly as it was produced. Due to its variability, plasma glucose concentration is clearly not a good indicator of stress on its own, but is still useful in  29  combination with other measurements such as plasma Cortisol due to the ease of measurement (Hardy and Audet 1990; Iwama et al. 1995). Phagocytosis was only affected by confinement and increased temperature which caused a significant reduction at 3 d. Superoxide production was also affected by the severe confinement, but, unlike phagocytosis, superoxide production was increased at 3 d. Increased superoxide production was also observed at one sampling point in 3 of the other experiments, however, the timing did not seem to be related to fish density, plasma Cortisol or plasma glucose concentrations. In the disease challenge it appears that the challenge dose was too high so that mortalities occurred too quickly to observe differences in mortality due to vibriosis as a result of stress. Also, it seems likely that confinement stress was not severe enough compared to the stress of the disease challenge. The disease challenge itself was a very stressful event and a few fish actually died from the stress of the challenge, and the confinement may have been minor in comparison. Differences in mortality due to tail rot were observed, and surprisingly were significantly higher in unconfined, shamchallenged fish, suggesting that confinement somehow offered protection against tail rot. This may be a result of stimulation of some aspect of immune function such as mucus lysozyme or continued stimulation of superoxide production observed in both shamchallenged groups. Increased lysozyme activity has been observed in Atlantic salmon (Salmo salar) and rainbow trout (O. mykiss) following stress (Fevolden et al. 1994; Mock and Peters 1990).  30  The mortality data could not be explained by differences in phagocyte function alone; however, some interesting observations were made. Superoxide production was significantly elevated in sham-challenged fish but not in pre-challenge or diseasechallenged fish. Similar results were reported by Angelidis et al. (1987) who found increased chemiluminescence in phagocytes from disease-challenged rainbow trout. They also observed decreased chemiluminescence and decreased disease resistance in stressed fish. Phagocytosis was significantly elevated in the unconfined disease-challenged fish, but not in the other groups. It is possible that this may have helped protect the fish and may have led to fewer mortalities at a lower challenge dose or with a different pathogen. Plasma Cortisol concentrations were elevated in both disease-challenged  groups  indicating, not surprisingly, that vibriosis is stressful. This is consistent with Robertson et al. (1987) who found that a disease outbreak evoked a  Cortisol  and glucose response in  red drum. Plasma glucose concentration was elevated in the unconfined, sham-challenged group, however because Cortisol was not elevated it is difficult to say what this might mean. These results do not indicate a period of decreased or impaired phagocyte function or a decrease in disease resistance resulting from confinement stress as would be expected from the results of other research with fish (Narnaware et al. 1994; Angelidis et al. 1987; Maule et al. 1989; Mazur and Iwama 1993; Wise et al. 1993). Decreased phagocytosis was observed at only one sampling point with the most severe stressor while increased superoxide production was observed at the same time and in several of the experiments. This is in contrast with the results of Narnaware et al. (1994) who found significantly  31  decreased phagocytosis in rainbow trout (O. mykiss) after 3 h of confinement. Pulsford et al. (1994), however, found increased phagocytic activity and increased peroxide production following transport stress in dab (Limanda limanda). Other researchers have shown enhancement and suppression of immune function due to stress in fish (reviewed in Weeks et al. 1992). In mammals, both suppression and enhancement of phagocyte function has been observed as a result of stress (reviewed in Zwilling 1994). The majority of the work on the effects of stress on immune function both in fish and in mammals has concentrated on specific immunity such as lymphocyte mitogenesis and antibody production. The majority of the results from these studies indicate that stress causes immune suppression, both in fish (Maule et al. 1989, Mazur and Iwama 1993) and in mammals (Keller et al. 1981), however, there are examples of enhanced specific immunity due to stress, both in fish (Maule et al. 1989) and in mammals (Jain and Stevenson 1991, Rinner et al. 1992, Lysle et al. 1990, Monjan and Collector 1977). Although stress is generally thought of as decreasing disease resistance, not all the available data supports this generalization (Cohen 1994, Pegg et al. 1995). There are examples of increased disease resistance resulting from stress in mammals (Moynihan et al. 1994, Hermann 1993) and in fish (Pegg et al. 1995) including the results presented here. Most work with fish so far indicates decreased disease resistance as a result of stress (Mazur and Iwama 1993, Maule et al. 1989, Angelidis et al. 1987, Peters et al. 1988), however, most disease challenge experiments do not represent realistic situations, especially injection challenges. Fish are subjected to huge doses of specific, known, highly virulent pathogens rather than small numbers of opportunistic microorganisms as  32  would likely be encountered in the wild. Disease challenge experiments are more relevant to aquaculture situations where large numbers of fish are kept in small spaces and sick and weak fish are not preyed upon so that large numbers of pathogens may be present during stressful situations. In cases where disease is allowed to spread and progress at a more natural rate results may be different. A n example of this is a disease challenge experiment performed on cutthroat trout (O. clarki) which had been disturbed daily by chasing with a net for several weeks (Pegg et al. 1995). Due to the low temperature at the time of this experiment the disease progressed very slowly and mortality was significantly higher in undisturbed fish. This difference in mortality may have been due to stimulation of some aspect of immune function such as the increase in superoxide production observed in the confinement experiments. These data add to the growing body of evidence that the relationship between stress, immune function, and disease resistance is not one of simple immunosuppression and decreased disease resistance (Zwilling 1994, Klein et al. 1992, Moynihan et al. 1994, Aarstad et al. 1991). Whether immune function is suppressed or enhanced depends on the type, severity, and duration of the stressor as well as the species and strain of animal, and the aspect of immune function measured (Laudenslager 1994, Moynihan et al. 1994, Rabin 1994). Both suppression and enhancement of different aspects of immune function have been observed in other studies with fish (Tahir et al. 1993, Thompson et al. 1993). In mammals not only are there examples of enhanced immune function with stress (Berkenbosch et al. 1991, Jain and Stevenson 1991, Coe et al. 1988, Wood et al. 1993, Millar et al. 1993) there are examples where stress had different effects on different  33  aspects of the immune system (Jessop et al. 1989, Sheridan et al. 1991, Shurin et al. 1994, Kusnecov and Rabin 1993, Okimura et al. 1986) or different effects on the same immune function at different times (Monjan 1977) or with different levels of stress (Rinnerera/. 1992). Since the stress response must be adaptive, the immune system is likely modulated in such a way as to best deal with stressful situations that occur in the wild. Most stressors in the wild, for example, would be acute stressors such as encounters with competitors or predators. The stress response causes changes that help the fish to deal with immediate problems such as energy requirement for fighting and escape. It has been estimated that responses to stress account for 25% of the fish's energy budget during such periods (Barton and Schreck 1987). Insults on the immune system during stress would result from cuts and abrasions which occur in such situations. Increased nonspecific immune function such as phagocytosis, superoxide production and lysozyme production would be more effective than specific immune function due to the long period of time required to mount specific immune responses. Temporarily decreased specific immune function would re-partition energy to more important processes in stressful situations. Specific immune function may be suppressed first to redirect energy and some aspects of nonspecific immune function may be stimulated, while in particularly stressful situations, both specific and nonspecific immune function may be suppressed. There are many fish diseases other than those commonly used in disease challenges and the changes in immune function that occur as a result of stress may not result in decreased resistance to all diseases. The changes that occur during stress may be  34  adapted to deal with external injuries that occur during fighting or escape and may result in increased resistance to diseases such as tail rot as observed in this study.  35  Chapter 2 Effects of Cortisol on Phagocyte Function in Juvenile Chinook (Oncorhynchus tshawytscha) and Coho Salmon (O. kisutch)  36  Introduction  Stress is experienced frequently by fish both in the wild and in aquaculture (Pickering 1989). Cortisol is the main corticosteroid produced in fish as a result of stress (Barton and Iwama 1991). Many researchers have reported suppression of immune function due to Cortisol. These include changes in the specific immune system such as decreased numbers of circulating lymphocytes (Pickering 1984), decreased numbers of antibody producing cells (Anderson et al. 1982; Slater and Schreck 1993), and decreased mitogen induced lymphocyte proliferation (Ellsaesser and Clem 1987;Grimm et al. 1985; Pulsford 1995). Changes also occur in the nonspecific immune system such as decreased chemiluminescent response in striped bass (Morone saxatilus) phagocytes (Stave and Roberson 1985) and decreased phagocytosis in dab (Limanda limanda) and channel catfish (Ictalurus punctatus) (Pulsford et al. 1995; Ainsworth et al. 1991b). Cortisol has also been shown to decrease disease resistance in fish (Houghton and Mathews 1986; Maule et al. 1987; Pickering 1989; Woo et al. 1987). Some of these studies, however, used very high or greater than physiological levels of Cortisol (Pulsford et al. 1995; Stave and Roberson 1985). Although Cortisol has been shown to decrease specific immune function and disease resistance, its effects on phagocyte function have not been clearly established. Narnaware et al. (1994) found no effect of Cortisol on phagocytosis at physiological concentration (80 ng/mL) in vitro with rainbow trout (Oncorhynchus mykiss) phagocytes. Pulsford et al.  (1994) also found no effect at 80 ng/mL in dab, but found a slight  37  stimulation of phagocytosis at 120 ng/mL and reduced phagocytosis at 320 ng/mL in vitro while no effect was observed in channel catfish from 50-1000 ng/mL (Ainsworth et al. 1991b). Most of the research on the effects of Cortisol in salmonids has been done with rainbow trout and coho salmon (O. kisutch) and little is known about the effects of Cortisol in the commercially cultured chinook salmon (O. tshawytscha). Cortisol is also produced during smolting for the transition to saltwater (Barton et al. 1985). Although there is some evidence that immune function and disease resistance may be affected (Maule et al. 1987), it seems unlikely that disease resistance is significantly compromised during smoltification. To determine the role of Cortisol in modulating phagocyte function, this series of experiments involved injecting or implanting Cortisol into fish. In vitro experiments were also conducted in which isolated phagocytes were incubated with media containing Cortisol or serum from stressed and control fish.  38  Materials and Methods  Fish: Juvenile chinook (Oncorhynchus tshawytscha) and coho salmon (O. kisutch) were obtained from commercial and federal hatcheries. Fish were maintained in dechlorinated Vancouver city water with the exception of the coho salmon used in the prednisolone injection experiment which were maintained in Cultus Lake water. Fish were fed a commercial salmon diet three to four times per week and acclimated for at least two weeks prior to each experiment. Cortisol Injection: Juvenile coho salmon (average 22 g) were injected with a Cortisol analog (prednisolone, 20 mg/g fish weight into the dorsal sinus) or saline (controls) and kept in 75 L aquariums. Four fish from each group were sampled after 2 h, and 2,4,7, and 14 d to measure phagocytosis. Isolated phagocytes were incubated in chamber slides with yeast for 2 h after which nonadherent cells and unphagocytized yeast were washed away. The slides were dried, stained with Diff Quik, and the percentage of phagocytes which contained yeast cells were counted under a microscope. Baseline data from control fish sampled in experiments from the previous 4 weeks is included for comparison, but not compared statistically since these fish were living in raceways rather than aquariums. Cortisol Implantation: Two Cortisol implantation experiments were performed. In each experiment, fish were implanted in the peritoneal cavity with Cortisol (hydrocortisone, Sigma) dissolved in a mixture of 50% coconut oil and 50% vegetable oil. Experiment 1) Juvenile coho salmon were implanted with Cortisol 100 mg/g fish  39  weight and sampled at 1 d and 5 d post-implantation. Experiment 2) Juvenile chinook salmon were implanted with two concentrations of Cortisol implants to give final doses of 50 and 100 mg/g fish weight (Specker et al. 1994). Control fish were injected with implants without Cortisol. Fish were kept in 70 L tanks and four fish per treatment were sampled after 1, 2, 3, 5, and 7 d to measure phagocyte function, plasma Cortisol concentration and glucose concentration. In vitro Cortisol: Phagocytes were isolated from juvenile chinook salmon and allowed to adhere to microplates as described above. Half of the wells from each fish _7 were incubated at 15°C with media containing 5x10 M (180 ng/mL) Cortisol (hydrocortisone 21-hemisuccinate, Sigma) and the other half were incubated in media without Cortisol. Phagocytes were incubated with Cortisol for 4, 8, 24, and 48 h at the end of which time phagocytosis, superoxide, and protein assays were performed. At 4, 8, and 24 h, four fish were used with duplicate wells for each assay, and at 48 h two fish were used with four replicate wells per assay. In vitro serum: Ten juvenile chinook salmon were transferred to 10 L of water in each of two white 25 L buckets with aeration and sampled at 1 h and 3 d. Blood samples were taken and allowed to clot at 4°C overnight before centrifuging to separate the serum. Control serum was collected from 10 fish from a 70 L tank. The serum was diluted to 10% in L-15 and frozen in aliquots at -50°C until use. Isolated phagocytes from four chinook salmon were incubated with either serum from stressed or control fish for 4 h before the media was removed and assays of phagocyte function were performed. In a separate experiment, pooled phagocytes from four fish were incubated for 2 h with serum  40  from stressed and control fish after which N B T was added without removing the media and incubated for another 2 h so that the serum was present during the assay. Protein was not measured since cells from the same population were used for all treatments so that standardization was not necessary. Phagocyte function: Phagocytes were isolated from the anterior kidney according to the method of Secombes (1990). A slide assay of phagocytosis was used in the Cortisol injection experiment in which the adherence and phagocytosis steps were performed at the same time. A slide assay of phagocytosis with separate adherence and phagocytosis steps was performed in the coho Cortisol implantation experiment and microplate assays were used for all other experiments  Table 2-1. Experimental conditions of the Cortisol experiments  Experiment  Time of Year  Water  Ave. fish  Temp.  weight  -  22 g  coho Cortisol injection  April  coho Cortisol implant  December  7°C  78 g  chinook Cortisol implant  April  7°C  29 g  in vitro Cortisol  June  11°C  35 g  in vitro serum  May  9°C  16 g  41  Statistical analyses: Student's t-tests were used to compare means for each day in the coho Cortisol implant and in vitro Cortisol experiments. Proportion data were arcsin square root transformed. In cases where data were not normally distributed, or variances were not equal, a Mann-Whitney U test was used. Two way analysis of variance was used for the Cortisol implantation experiment. One way A N O V A was used to compare treatments in the in vitro serum experiment and Cortisol injection. Student-NewmanKeuls tests were used to determine group differences. Significance level for all experiments was p<0.05 and all data are presented as means +/- standard errors. A l l statistical analysis was done using SigmaStat statistical software (Jandel Scientific, San Rafael, California, USA).  42  Results  Cortisol injection: Initial suppression of phagocytic activity was seen both in the Cortisol and saline injected at 2 h and 2 d, however, by day 4 saline injected coho had returned to baseline while Cortisol injected fish showed significantly lower phagocytic activity on days 4,7, and 14 (Figure 2-1). Cortisol implantation: Cortisol implantation caused a slight, but insignificant decrease in percent phagocytosis in juvenile coho salmon at both 1 and 5 d (Figure 2-2). In both groups of chinook salmon implanted with Cortisol, plasma Cortisol concentrations remained significantly elevated over controls in the high physiological stress range for the duration of the experiment (Figure 2-3A). Cortisol implantation raised plasma Cortisol concentrations to between 395 and 507 ng/mL for the 100 mg/g dose and between 173 and 431 ng/mL for the 50 mg/g dose, compared to between 15 and 56 ng/mL for sham implanted fish. Plasma glucose concentration was higher in both groups of Cortisol implanted fish than sham injected fish overall, however, there was no difference between the two doses, or on individual days (Figure 2-3B).Two way analysis of variance showed a significant effect of both time and dose on phagocytosis. Overall, Cortisol implanted fish showed significantly higher phagocytosis than sham injected fish, however, there was no difference between the two doses, or on individual days (Figure 2-3C). Superoxide production was not significantly different in Cortisol implanted fish than in sham implanted fish (Figure 2-3D).  43  In vitro Cortisol: Cortisol had no effect on phagocytosis or superoxide production in vitro at high physiological concentration, although both phagocytosis and superoxide production seemed to increase with time (Figure 2-4). In vitro  serum: Phagocytes incubated for 4 h with 10% serum from 1 h and 3 d  stressed fish showed significantly reduced phagocytosis (Figure 2-5A) but no effect on superoxide production (Figure 2-5B). When pooled phagocytes were incubated for 2 h and the media containing serum was not removed increased superoxide production was observed (Figure 2-5 C). Media Cortisol concentration was 26 ng/mL for the 1 h stressed serum treatment, 25 ng/mL for the 3 d stressed serum treatment, and 6 ng/mL for the control serum treatment.  44  Figure 2-1. The effects of a single Cortisol injection (prednisolone 20mg/g) in the dorsal sinus of juvenile coho salmon, Oncorhynchus kisutch, on percent phagocytosis of glass adherent phagocytes isolated from the anterior kidney. Means (+/- 1 S.E.), N=4, * p<0.05. Baseline is control fish from previous experiments maintained in raceways rather than in aquaria.  45  ] control  1 day  5 days  ure 2-2. The effects of Cortisol implantation (lOOug/g) on percent phagocytosis isolated from juvenile coho salmon, Oncorhynchus kisutch. Means (+/- 1 S.E.), N=4, * p<0.05.  46  Figure 2-3. The effects of two doses of Cortisol implants (50 and 100 mg/g) and sham implants on plasma Cortisol concentration(A), plasma glucose concentration(B), phagocytosis(C), and superoxide production (D) of adherent anterior kidney phagocytes isolated from juvenile chinook salmon, Oncorhynchus tshawytscha, Means (+/- 1 S.E.), N=4, * both doses different from control, ** both doses different from control and from each other( 2-way A N O V A p<0.05).  47  o  o l_  0.  D) </>  re  a>  4  8  24  48  Time (hours)  Figure 2-4. In vitro effects of Cortisol (181ng/mL / 5x10" M ) on phagocytosis and superoxide production in phagocytes isolated from juvenile chinook salmon, Oncorhynchus tshawytscha. Means (+/- 1 S.E.), N=4, * p<0.05.  48  ra v >-  CQ  Z O)  H CO  z o>  Figure 2-5. In vitro effects of 10% serum from juvenile chinook stressed for 1 h or 3d on phagocytosis (A), superoxide production without serum present (B), and superoxide with serum present (C) of phagocytes isolated from juvenile chinook salmon, Oncorhynchus tshawytscha. Means (+/-1 S.E.), N=4, * p<0.05.  49  Discussion  Cortisol injection and Cortisol implantation in coho salmon caused decreases in phagocytosis (although not statistically significant in the Cortisol implant experiment) measured using a slide assay. Cortisol implantation in chinook salmon, however, showed increased phagocytosis. There are several possible reasons for this observed difference including: the methods used to measure phagocytosis; the dose, age of fish, time of year and species and strain differences. There is evidence for species differences in sensitivity to physiological concentrations of Cortisol in fish (Pickering et al. 1989). This might explain the differences in the two Cortisol implant experiments while the difference between the Cortisol implant experiments and the prednisolone injection experiment may be due to the much higher dose used in the prednisolone injection experiment (20 mg/g compared to 50 ug/g and 100 u.g/g in the Cortisol implant experiments). There is also evidence for seasonal changes, in sensitivity of immune function to Cortisol in coho salmon (Maule et al. 1993) and although these experiments were performed at approximately the same time of year there may be species differences in the timing of changes in Cortisol sensitivity. The two phagocytosis assays used measure phagocytosis slightly differently. The slide assay of phagocytosis measures the percentage of phagocytes that are actively engulfing yeast cells (the number of yeast per cell can also be measured) while the microplate assay measures the total amount of yeast cells engulfed by a population of phagocytes, but does not measure the proportion of phagocytes that have engulfed yeast  50  cells or the number of yeast cells engulfed per phagocyte. It is possible that although fewer phagocytes are actively engulfing yeast cells, they are engulfing more yeast per phagocyte so that the overall number of yeast engulfed is higher. These results indicate that Cortisol does affect phagocytosis when administered in vivo, but had no effect on superoxide production in chinook salmon. The results demonstrate the importance of choosing the appropriate method of measuring phagocyte function and the problems with interpreting and extrapolating the results of one assay of immune function to immune competence and disease resistance. The lack of effects of Cortisol in vitro indicate that the effects of Cortisol on phagocyte function are probably not direct. A similar lack of response of phagocytes to physiological levels of Cortisol in vitro has been reported in rainbow trout at 80 ng/mL (Narnaware et al. 1994) and in channel catfish from 50-1000 ng/mL (Ainsworth et al. 1991b). Pulsford et al. (1995) found no effect on phagocytosis in vitro from 80-120 ng/mL, but found a significant reduction of phagocytosis at 320 ng/mL in dab. Stave and Roberson (1985) also found a reduction of the chemiluminescent response of striped bass phagocytes at pharmacologic concentrations of Cortisol. In vitro incubation of phagocytes from unstressed fish with serum from stressed fish resulted in decreased phagocytosis and increased superoxide production which is similar to the results observed with severe confinement stress (see chapter 1). This change in superoxide production was only observed in the presence of the serum which is consistent with the results of DeKoning and Kaattari (1991) who found that fish lymphocytes were more responsive when incubated with homologous serum. Similar  51  experiments have been performed in both fish (Alford, 1994) and in rodents (Zha et al. 1992) in which incubating cells from unstressed animals with serum from stressed animals was able to mimic the effects of stress. The final Cortisol concentration of the media in the in vitro serum experiments was only 25 ng/mL which is much lower than the 180 ng/mL added in the in vitro Cortisol experiment. This suggests that something in the serum other than or in addition to Cortisol is affecting phagocyte function. This adds to the evidence collected by other authors that indicates that in fish the effects of Cortisol on the immune system are not direct and that other factors are involved (Narnaware et al. 1994; Maule et al. 1993; Ainsworth et al. 1991b; Maule and Schreck 1990; Ellsaesser and Clem 1987; Secombes et al. 1996). Other researchers have also shown differences between in vitro and in vivo effects of Cortisol. Ainsworth et al. (1991b) found a significant reduction of phagocytosis after injection of Cortisol in channel catfish, but found no effect of stress resulting in elevated plasma Cortisol concentrations, and no effect of Cortisol in vitro. There is also evidence that Cortisol may affect parts of the immune system differently. Pulsford et al. (1995) found that phagocytosis in dab was only affected at 320 ng/mL, the highest dose tested, but lymphocyte proliferation was affected in a dose dependent manner from 80-320 ng/mL. Specific immune function may be suppressed in a dose dependent manner while phagocyte function is only suppressed beyond a threshold concentration (Pulsford et al. 1995). Differences between the results presented here and by other researchers indicate that there may be species differences in phagocyte responses to Cortisol, at least in the concentration required to get a response (Pulsford et al. 1995; Ainsworth et al. 1991b).  52  Research with mammals shows similar results. Although Cortisol is generally considered to be immunosuppressive (Munck et al. 1984) there is evidence that increased corticosteroid levels is not the main mechanism of stress-induced changes in immune function (Pezzone et al. 1992, Klein et al. 1992, Zha et al. 1992, Flores et al. 1990, Wiegers et al. 1994, Keller et al. 1983, Ader et al. 1987). A variety of effects, ranging from suppression to enhancement, on different aspects of phagocyte function (reviewed in Zwilling 1994 and in Cupps and Fauci 1982) and other aspects of immune function (reviewed in Ader et al. 1987) have been observed in mammals. The results presented here combined with the results of other authors indicate that the effects of Cortisol on immune function both in fish and in mammals is not one of simple suppression. The effects of Cortisol on immune function may depend on the dose of Cortisol used, age and life stage of the fish, species differences, and the aspect of immune function measured.  53  Chapter 3 The Effects of Pulp M i l l Effluent Exposure on Phagocyte Function in Juvenile Chinook Salmon (Oncorhynchus tshawytscha)  54  Introduction  The upper Fraser River is an important rearing area for juvenile chinook salmon (Oncorhynchus tshawytscha) (Rosberg and Associates 1987). There are four bleached kraft pulp mills on the upper Fraser River near Prince George and Quesnel and the concentration of biotreated bleached kraft pulp mill effluent (TBKME) in the river at winter flows has been estimated at 1.5-3% (Servizi et al. 1993). M i l l effluent is tested to ensure that it is not acutely toxic and it has been assumed by regulatory bodies that this would assure that the aquatic environment is safe for fish; however, residual organochlorines have been detected in both T B K M E and juvenile chinook salmon in the upper Fraser River (Servizi et al. 1993). There may be sublethal effects of chronic exposure that cannot be detected or predicted by acute toxicity testing. In addition, the effects of T B K M E are difficult to predict due to its complex nature and site specific factors (Owens 1991). The immune system is a highly complex network of cells which are capable of rapid responses including proliferation and differentiation and so is vulnerable to contaminants (Weeks et al. 1992). Changes in immune function can provide sensitive early warning indicators of toxicity (Rice et al. 1996; Weeks et al. 1992; Mathews et al. 1990) and link the increased incidence of disease with contaminants in highly polluted waters (Weeks and Warinner 1984). Nonspecific immunity is one of the first lines of defense against invading microorganisms in fish and is thought to be more important than specific immunity at low  55  temperatures and during the early life stages (Ainsworth et al. 1991a; Chen and Ainsworth 1991; O'Neil 1985). This may be important for fish in the Fraser River since temperatures can be less than 1°C for extended periods of time in the winter months (Emmett et al. 1996). There is also some evidence that contaminants such as polychlorinated biphenyl (PCB) may affect nonspecific immune function more than specific immune function (Rice and Schlenk 1995). There is evidence that fish do not avoid T B K M E (Swanson et al. 1994). In fact, in a survey of the mixing zone of the mill in this study, more fish were captured in the mixing zone than in an upstream reference site suggesting that the fish may prefer the mixing zone due to its warmer temperature (Emmett et al. 1996; ). In addition, 90% of the fish captured in the mixing zone were chinook salmon (Emmett et al. 1996). Ice cover in the winter combined with oxygen demand of T B K M E may lead to significantly reduced oxygen concentrations in the river (Emmett et al. 1996). Many studies have demonstrated negative effects of contaminants on immune function in fish (Anderson et al. 1989; Rice and Weeks 1989; Bennet and Wolke 1987a, 1987b; Walczak et al. 1987) including nonspecific immune function (Rice et al. 1996; Voccia et al. 1994a; Seeley and Weeks-Perkins 1991; Warinner et al. 1988; Thuvander et al. 1987; Weeks et al. 1986, 1987a, 1987b; Weeks and Warinner 1984, 1986; Rice and Schlenk 1995). However, relatively little work has been done on the effects of T B K M E on immune function in fish (Voccia et al. 1994b) or on the effects of contaminants on immune function in chinook salmon (Arkoosh et al. 1994). Recently, Voccia et al.  56  (1994b) have shown that T B K M E suppresses phagocytosis and stimulates mitogenesis in vitro at 8.75 to 35% in rainbow trout (Oncorhynchus mykiss).  In this study juvenile chinook salmon were exposed in situ to T B K M E for 30 d under both normoxic and hypoxic conditions at concentrations relevant to those found in the river during winter months. Phagocytosis and superoxide production of anterior kidney phagocytes were measured to determine whether T B K M E affects immune function in chinook salmon.  57  Materials and Methods  TBKME Exposure: Juvenile chinook salmon were exposed to 16%, 8%, 4%, 2%, and 0% (control) pulp mill effluent for 30 d under both normoxic and hypoxic (65% +/-1% oxygen saturation) conditions. The T B K M E exposure experiments were performed in a portable lab at the Northwood Pulp M i l l near Prince George, B.C., Canada. Fish were maintained in water pumped directly from the Fraser River. Pulp mill effluent was pumped directly from the final treatment pond of the Northwood Pulp M i l l , cooled from 20 - 25°C to 15 - 17°C, and mixed to give the above concentrations. There was a slight temperature gradient due to the warmer temperature of the pulp mill effluent from 1°C in the control tanks to 3 - 3.5°C in the 16% pulp mill effluent tanks. Three experiments were performed: 1) a hypoxic experiment in 200 L flowthrough tanks with both normoxic and hypoxic control groups; 2) a normoxic experiment in 200 L flow-through tanks; and 3) a normoxic experiment in 70 L flow through donutshaped tanks in which water was circulated with a pump to create a current. In the hypoxic experiment oxygen concentration was reduced to 65% +/- 1% of saturation using a vacuum degasser. In the normoxic experiments oxygen concentration was 80-85% of saturation. In experiment 3) the bottoms of the donut-shaped tanks were lined with river stones and a pump recirculated the water to create a current of approximately 1.5 body lengths per second. Flow was 10 L/min in the 200 L tanks and 2 L/min in the 70 L donutshaped tanks. Total number of fish in each 200 L tank was 80 and 60 in the 70 L donut tanks. Fish density in the 20 L tanks was 3.6 g/L and 2-3 times higher in the donut tanks.  58  Due to time constraints, not all treatments were sampled for phagocyte function. In experiment 1) phagocyte function was measured in the 16%, 4% group, and both control groups, in experiment 3) phagocyte function was measured in the 16%, 4%, 2% and control group, and in experiment 2) phagocyte function was measured in all treatments. Experiments were performed from January to March 1996. Phagocyte Isolation: Anterior kidneys were removed from eight fish per treatment and placed in separate tubes containing 2 mL of Leibovitz (L-15) medium supplemented with 10 u/mL heparin and antibiotics on ice. Cell suspensions were created by teasing the tissue through 54 um nylon mesh. The cell suspensions were layered onto a 34%/51% discontinuous percoll gradient and centrifuged for 20 min at 400 x G (Secombes 1990). The phagocyte-rich cell suspension was collected from the interface, washed once in 5 mL L-15, and resuspended in 0.25 mL L-15 supplemented with 0.1% fetal calf serum (L-15+FCS). Assays of Phagocyte Function: Three slides were prepared for phagocytosis assays using 10%, 50%, and 100% cell suspension in lOOuL L-15+FCS. Duplicate slides were prepared for the nitro blue tetrazolium (NBT) reduction assay of superoxide production with 50 uL cell suspension per well. Slides were incubated at 10°C for 30 min in a humid chamber to allow the cells to adhere after which time the nonadherent cells were washed away with a gentle stream of phosphate buffered saline (PBS). For the phagocytosis assay 100 uL of 2x10 autoclaved, washed yeast in L-15+FCS was added to each well. For the N B T reduction assay 2 mg/mL N B T in 0.85% saline was added to each well. The slides were incubated for 2 h at 10°C in a humid chamber. The  59  phagocytosis slides were rinsed with a gentle stream of P B S to remove any unphagocytized yeast and then fixed in methanol for 10 min. The phagocytosis slides were later stained with either giemsa or Diff Quick and examined under a compound microscope to determine the percentage of phagocytes which had engulfed one or more yeast cells. The N B T slides were examined immediately under a compound microscope to determine the percentage of cells which had reduced the N B T and produced a blue colour (NBT positive). Plasma Cortisol and Glucose Concentration: Plasma samples were collected from fish in the normoxic tub experiment in hematocrit tubes and frozen on dry ice until it was analyzed for glucose using a microplate adaptation of the Trinder method (Sigma) and for Cortisol using a radioimmune assay (Diagnostics Product Corporation). 10 uL of plasma was used for the Cortisol rather than 25 uL due to the small volume of plasma collected. Statistical Analyses: Group means were compared using one way analysis of variance after arcsin square-root transforming the proportion data and the StudentNewman-Keuls Method was used to determine which groups were different. A Pearson product moment correlation was done on the plasma glucose data. Significance level for all experiments was p<0.05 and all data are presented as means +/- standard errors, N=8. A l l statistical analysis was done using SigmaStat statistical software (Jandel Scientific, San Rafael, California, USA).  60  Results  Experiment 1) hypoxic tanks: The number of N B T positive cells was significantly elevated in the hypoxic control group compared to the normoxic control group (Figure 3-1 A). The number of N B T positive cells was also significantly elevated in the 4% group compared to the normoxic control group, but only about half as much as the hypoxic control group (Figure 3-1 A). The number of N B T positive cells in the 16% group was not significantly different from the normoxic control group (Figure 3-1A). Phagocytosis was not significantly different in the hypoxic control fish compared to the normoxic control fish, however, phagocytosis was lower in both of these control groups compared to the control groups from the two normoxic experiments (Figure 3-IB). Under hypoxic conditions phagocytosis was significantly reduced in the 16% group, but not in the 4% group (Figure 3-1B). There was no difference in mean fish weight between treatments (9.5 +/- 0.32 g). Experiment 2) normoxic tanks: The number of N B T positive cells was significantly increased in the 2% and 4% groups but not in the 8% and 16% groups. (Figure 3-2A). Phagocytosis was significantly reduced in the 2% and 4% groups and not in the 8% and 16% groups (Figure 3-2B). Plasma Cortisol concentration was significantly lower than controls in the 2% and 16% groups but not different in the 4% and 8% groups (Figure 3-3A). Plasma glucose concentrations decreased with increasing pulp mill effluent concentration and were significantly lower than controls in the 4%, 8%, and 16% groups (Figure 3-3B). There was a significant correlation between plasma glucose  61  concentration and pulp mill effluent concentration (R2=0.61)(Figure 3-3B). There was no difference in mean fish weight between treatments (9.6 +/- 0.27 g). Experiment 3) normoxic donut-shaped tanks: The number of N B T positive cells was significantly increased in the 2% and 4% groups but not in the 16% group. (Figure 3-2A). Phagocytosis was significantly reduced at all three concentrations of pulp mill effluent  (Figure 3-2B). There was no difference in mean fish weight between  treatments (8.8 +/- 0.28 g).  62  ;ure 3-1. Effects of 30 day exposure to several concentrations of pulp mill effluent and hypoxia on superoxide production (A) and phagocytosis (B) of anterior kidney phagocytes from juvenile chinook salmon (Oncorhynchus tshawytscha) Means (+/-1 S.E.), N=8, * different from normoxic control at p<0.05.  63  40 O  30  > '55  o °- 20 S  10 oH  90 H in  Io 80 o  O)  5 70 Q. c 0)  -••  60  donuts tubs  0.  50  o  n 2  r 4  8  16  Percent Pulp Mill Effluent  gure 3-2. Effects of 30 day exposure to several concentrations of pulp mill effluent on superoxide production (A) and phagocytosis (B) of anterior kidney phagocytes from juvenile chinook salmon (Oncorhynchus tshawytscha) under normoxic conditions. Identical experiments were performed in 70 L donut-shaped tanks (donuts) and 170 L tub-shaped tanks (tubs). Means (+/-1 S.E.), N=8, * p=0.05.  64  90 80  H  E 70 o>  r  60 H 50 40 H 30 20 H 10  B  70  *  T 60 H  *  50 -A  *  40 -A  30 H  0  2  4  8  16  % Pulp Mill Effluent  gure 3-3. Effects of 30 d exposure to several concentrations of pulp mill effluent on plasma Cortisol and glucose concentrations of juvenile chinook salmon (Oncorhynchus tshawytscha) under normoxic conditions. Means (+/- 1 S.E.), N=8, * p=0.05.  65  Discussion  Hypoxia had a significant effect on the number of NBT-positive cells in the absence of pulp mill effluent as well as in the presence of low (4%) concentration of pulp mill effluent, however, this effect was higher under normoxic conditions. This suggests that there is an interaction between hypoxia and pulp mill effluent. It is possible that the hypoxic conditions decreased the ability of the immune system to respond to the pulp mill effluent, or vice versa. Conversely, phagocytosis was only reduced at the highest concentration of pulp mill effluent under hypoxic conditions. The largest differences in both phagocytosis and N B T reduction were observed in the 2 and 4% groups. Phagocytosis was suppressed at the lower concentrations as well as in the 16% group in the normoxic tank experiment while N B T reduction was stimulated at the lower concentrations. Anderson (1992) describes the increase in NBT-positive cells is an indication of an alarm reaction such as one produced by an immunogen. The lack of effects at the higher concentrations may indicate that the system is exhausted and unable to respond or that the system has returned to normal. At lower concentrations there may be a prolonged alarm reaction. Changes detected in these assays indicate that pulp mill effluent does have an effect on the immune systems of the fish exposed at low concentrations. Any change in the immune system has the potential for compromising the fishes resistance to disease. The fact that the greatest effects were seen at the lowest concentrations is important since these are the concentrations that the fish are more likely to encounter in the wild.  66  A similar pattern of responses has been observed in other fish exposed to contaminants. Hematocrit values and numbers of lymphocytes in dab {Limanda limanda) were increased at low concentrations of oil contaminated sediment, but decreased at high concentrations (Tahir et al. 1993). The chemiluminescent response of oyster toadfish was found to be increased at low concentrations of tributyltin and returned to baseline at higher concentrations (Rice and Weeks 1989). Seeley and Weeks-Perkins (1991) found both increased and decreased phagocytosis in fish from sites with different levels of contaminants compared to a reference site. Several other studies have also shown changes in immune function due to contaminants (Weeks and Warinnner 1984, 1986; Rice et al. 1996; Weeks et al. 1986; Anderson et al. 1989; Rice and Schlenk 1995). The Cortisol data does not seem to be related to T B K M E concentration, plasma glucose concentration, phagocyte function, sampling order or time of day. The glucose data showed a significant T K B M E concentration dependence. Since the fish were fed during the experiment and the fish in the higher T B K M E concentrations had not lost weight compared to the control fish, it is possible that the decrease in plasma glucose concentrations is due to increased clearance of glucose as a result of the increased metabolic demand of detoxifying the T B K M E .  67  Concluding Remarks  The results presented in this thesis indicate that stress and Cortisol do not have a general immunosuppressive effect. Both stimulatory and suppressive effects of stress, Cortisol and pulp mill effluent exposure on phagocyte function were observed. It was also shown that stress does not always result in decreased disease resistance. This adds to the growing body of evidence, both in fish and in mammals, that stress-induced immunomodulation is more complex than general immunosuppression and decreased disease resistance mediated by elevated corticosteroid levels. Although stress certainly can suppress immune function and decrease disease resistance, this is not always the case. It has been shown here that stress can not only stimulate immune function, in some cases it can also have different effects on different parts of the immune system. Whether immune function is suppressed or enhanced may depend the type, severity and duration of the stressor, as well as which aspect of immune function is measured. Since different parts of immune function can be affected differently, it is desirable to measure as many different aspects of immune function as possible. There is also little doubt that corticosteroids can also suppress immune function, especially in high physiologic and pharmacologic doses. There is growing evidence, however, that elevated corticosteroid levels may not be the main mechanism of stressinduced changes in immune function. In this thesis, the effects of Cortisol on phagocyte function ranged from suppressive to stimulatory. Also, in the pulp mill effluent  68  experiments, both stimulation and suppression of phagocyte function was observed without elevations in plasma Cortisol concentrations. Whether disease resistance is reduced or enhanced as a result of stress will depend on the changes in immune function which occur as well as the type of disease agent, dose, and timing of exposure. Diseases that are common in aquaculture may be the ones that are able to take advantage of the changes in immune function that occur as a result of stressors common in aquacultural practices. Resistance to some diseases, such as those caused by opportunistic infections like tail rot, may be increased by the changes in immune function that occur during stress. The effects of pulp mill effluent, which can be considered a stressor, on phagocyte function were also complex. Both stimulatory and suppressive effects were observed at low concentrations and almost no effect was observed at higher concentrations. Although it is difficult to say for certain whether disease resistance may be compromised since both stimulation and suppression of phagocyte function were observed, any change in phagocyte function has the potential to affect disease resistance, especially in light of the importance of phagocytes at low environmental temperatures such as those experienced by juvenile chinook overwintering in the upper Fraser River. The relationship between stress, immune function, and disease resistance is complex. There are many types and degrees of stressors and contaminants and each may have different effects on the immune system and disease resistance. More research is needed. If a stressor such as handling or transport does indeed decrease some aspects of immune function, such as antibody production, an appropriate immunostimulant may be  69  administered to counteract these effects. If an immunosuppressive effect is known to last for a certain period of time, fish can be allowed to recover and monitored more closely so that any disease problems are detected early.  70  Future Research This study raises more questions than it answers. One question that needs to be answered is how the different types of assays of phagocyte function compare with each other. As discussed earlier there are a large number of different assays of phagocyte function and there has been little comparison between them. It would be very useful to do an experiment in which a variety of assay techniques are compared in their ability to detect a known imrnunomodulator. This type of information would be useful to compare research done using different techniques. The in vitro serum experiment may be useful as an in vitro model for testing serum samples for the presence of immunomodulating substances. The same type of confinement stress experiments could be performed, but serum samples could be collected instead of phagocytes. These serum samples could all be tested at the same time for their ability to cause changes in pooled phagocytes from other fish. This type of experiment would eliminate a large amount of variation between fish and days as well as the time required for isolating cells from individual fish and adjusting cell concentrations. This type of experiment would also be useful for environmental monitoring. Serum samples could be collected from fish in the field where immune assays would not be possible and transported back to the lab for testing. Future experiments should also be designed to measure as many different aspects of immune function as possible, including both specific and nonspecific immune function. Since even different functions of the same type of cell can be affected  71  differently by different treatments, i f only one or a few immune functions are measured you are not getting the whole picture. It would be interesting to compare a variety of types and degrees of stressors to see which ones affect phagocyte function. In order to determine the timing and duration of modulation of phagocyte function during stress it would be useful to chose a stressor that has a large effect on phagocyte function since it is difficult to process large numbers of samples for assays of phagocyte function and subtle effects are easily missed. The disease challenge experiment raised some interesting questions. Modulations in immune function as a result of stress are undoubtedly adaptive, however, several researchers have shown decreases in disease resistance due to stress. In this experiment, however, an increase in disease resistance was observed in confined fish. 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