UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

Effects of adjuvanted AEROMONAS SALMONICIDA vaccines on growth, oxygen consumption, and selected haematological… Ackerman, Paige A. 1995

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Item Metadata

Download

Media
831-ubc_1995-0199.pdf [ 4.26MB ]
Metadata
JSON: 831-1.0086740.json
JSON-LD: 831-1.0086740-ld.json
RDF/XML (Pretty): 831-1.0086740-rdf.xml
RDF/JSON: 831-1.0086740-rdf.json
Turtle: 831-1.0086740-turtle.txt
N-Triples: 831-1.0086740-rdf-ntriples.txt
Original Record: 831-1.0086740-source.json
Full Text
831-1.0086740-fulltext.txt
Citation
831-1.0086740.ris

Full Text

EFFECTS OF ADJUVANTED  AEROMONAS SALMONICIDA VACCINES  OXYGEN CONSUMPTION, AND SELECTED HAEMATOLOGICAL AND IMMUNOLOGICAL VARIABLES IN JUVENILE RAINBOW TROUT  (ONCORHYNCHUS MYKISS). by PAIGE A. ACKERMAN B.Sc, The University of British Columbia, 1991  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Department of Animal Science) We accept this thesis as conforming to the required standard  "nrfe UNIVERSITY OF BRITISH COLUMBIA April 1995 © Paige Adrienne Ackerman, 1995  ON GROWTH,  In  presenting  degree freely  at  this  the  available  copying  of  department publication  of  in  partial  fulfilment  of  the  University  of  British  Columbia,  I  agree  for  this or  thesis  reference  thesis by  this  for  his thesis  and  scholarly  or for  her  Department  DE-6  (2/88)  Columbia  I  further  purposes  gain  shall  that  agree  may  representatives.  financial  permission.  T h e U n i v e r s i t y o f British Vancouver, Canada  study.  requirements  It not  be is  that  the  Library  an  granted  by  allowed  advanced  shall  permission  understood be  for  the that  without  for head  make  it  extensive of  my  copying  or  my  written  Abstract Seven injectible vaccines against Aeromonas  salmonicida  were tested to determine  the various effects they might have on selected variables of the physiology, swimming performance and immune functions of juvenile rainbow trout (Oncorhynchus  mykiss).  The  study involved three trials of consecutively longer times, 5, 12, and 24 weeks in duration, at the beginning of which fish were injected with a variety of adjuvanted and non-adjuvanted vaccines. The array of vaccine preparations was based on a simple, formalin killed, Aeromonas  salmonicida  bacterin.  They were: bacterin only, bacterin adjuvanted with  levamisole, bacterin in oil emulsion, microencapsulated- bacterin, microencapsulated bacterin with the addition of muramyl dipeptide, microencapsulated bacterin with P-1,3 glucan added, and microencapsulated bacterin with the addition of Vibrio  anguillarum  lipopolysaccharide (LPS).  Midway through each of the three trials, oxygen consumption was determined in whole fish as an indicator of metabolic rate. At the termination of each study, fish weights and lengths were determined and specific growth rates were calculated.  Fish were  sacrificed and blood was collected for determination of haematocrit, leucocrit, differential white blood cell count, agglutinating antibody titre, and haemolytic activity in the plasma. Somatic organs were dissected from the animals, and the somatic indices for total kidney, head kidney and spleen were determined. Finally, kidney lysozyme activity was determined from tissue homogenates.  The highest level and broadest range of positive responses were gained from the A. salmonicida  bacterin which was microencapsulated with V. anguillarum  ii  LPS as an  immunostimulant. This preparation proved to increase the metabolic and specific growth rates offishover a short period of time. Such effects may have potential long term positive impacts on the size of fish at harvest. The LPS treatment also caused a high plasma antibody titre, and high levels of kidney lysozyme activity for a substantially longer period of time than other immunizations.  iii  Table of Contents Abstract Table of Contents List of Tables List of Figures Acknowledgments  ii iv vi viii x  GENERAL INTRODUCTION  1  CHAPTER 1. Effects of Adjuvanted Aeromonas salmonicida Vaccines on Growth and Oxygen Consumption Rate of Juvenile Rainbow Trout (Oncorhynchus  mykiss)  9  INTRODUCTION MATERIALS AND METHODS Fish Stocks and Vaccines Respirometry Growth Experiments Statistical Analyses RESULTS Growth and Condition Oxygen Consumption Rate DISCUSSION  10 11 11 13 16 17 18 18 18 21  CHAPTER 2. Some Immunological and Haematological Effects of Adjuvanted Aeromonas salmonicidaVaccines on Juvenile Rainbow Trout (Oncorhychus  mykiss)  26  INTRODUCTION MATERIALS AND METHODS Fish Stocks and Vaccines Blood Collection Haematocrit Measurement Leucocrit Measurement Differential Blood Cell Counts Measurement of Somatic Indices of Haematopoietic Organs Measurement of Kidney Lysozyme Activity Antigen Preparation Measurement of Agglutinating Antibody Titres Production of Sensitized Rabbit Red Blood Cells Measurement of the Haemolytic Activity of Plasma Statistical Analyses RESULTS Haematocrit Leucocrit Differential White Blood Cell Counts Somatic Indices of Haematopoietic Organs  IV  27 31 31 31 31 32 32 32 33 34 35 35 36 37 38 38 38 38 42  Table of Contents Kidney Lysozyme Activity Agglutinating Antibody Titres..... Haemolytic Activity of Plasma DISCUSSION  43 46 47 51  CONCLUDING REMARKS  64  REFERENCES  69  APPENDIX Data tables  82 82  v  List of Tables Table 1.  Experimental conditions for juvenile rainbow trout in three time trials  12  Table 2.  Vaccine Preparations  Table 3.  Projected final weights of rainbow trout after a two year growth cycle. Values are computed from The University of British Columbia Aquaculture Production Analysis Computer Program, (Iwama and Fidler, 1989) and based on a mean initial weight of 15 g .....22  Table 4.  Summarized significant results among seven immunizations over three time trials  .....13  68  Table 5.  Mean (± 1 SE) condition factors of juvenile rainbow trout at three different times following immunization with seven different A. salmonicida vaccines ....83  Table 6.  Mean (+ 1 SE) specific growth rates of juvenile rainbow trout at three different times following immunization with seven different A. salmonicida vaccines ....84  Table 7.  Mean (± 1 SE) oxygen consumption rates of juvenile rainbow trout at three different times following immunization with seven different A. salmonicida vaccines  85  Table 8.  Mean (± 1 SE) haematocrit values (% RBC) in juvenile rainbow trout blood at three different times following immunization with seven different A. salmonicida vaccines 86  Table 9.  Mean (± 1 SE) leucocrit values and total leucocytes in juvenile rainbow trout blood at three different times following immunization with seven different A. salmonicida vaccines 87  Table 10.  Mean (± 1 SE) percentage of lymphocytes and neutrophils in juvenile ; rainbow trout blood at three different times following immunization with seven different A. salmonicida vaccines 88  Table 11.  Mean (± 1 SE) percentage of thrombocytes and monocytes in juvenile rainbow trout blood at three different times following immunization with seven different A salmonicida vaccines 89  Table 12.  Mean (+ 1 SE) somatic index of the head kidney and total kidney in juvenile rainbow trout at three different times following immunization with seven different A. salmonicida vaccines  vi  90  Table 13.  Mean (± 1 SE) somatic index of spleen tissue in juvenile rainbow trout at three different times following immunization with seven different A. salmonicidavaccines  Table 14.  Table 15.  Table 16.  91  Mean (+ 1 SE) kidney lysozyme activity in juvenile rainbow trout at three different times following immunization with seven different A. salmonicida vaccines. Values are expressed as units of activity per gram kidney tissue Mean (± 1 SE) agglutinating antibody titres in juvenile rainbow trout at three different times following immunization with seven different A. salmonicida vaccines. Values are expressed as the reciprocal of the highest agglutinating dilution  ....92  93  Mean (± 1 SE) plasma haemolytic activity in juvenile rainbow trout at three different times following immunization with seven different Aeromonas salmonicida vaccines 94  vii  List of Figures Figure 1.  Figure 2.  Figure 3.  Specific growth rates of immunized juvenile rainbow trout over three time trials. Means (± 1 SE) with different superscripts differ significantly (P < 0.05) by all pairwise multiple comparison Oxygen consumption rates of immunized juvenile rainbow trout over three time trials. Means (± 1 SE) with different superscripts differ significantly (P < 0.05) by all pairwise multiple comparison Overview of complement activation pathways. The classical pathway is initiated by C1 binding to antigen-antibody complexes, and the alternative pathway is initiated by C3b binding to various activating surfaces, such as microbial cell walls. The C3b involved in alternative pathway initiation may be generated in several ways, including spontaneously, by the classical pathway, pr by the alternative pathway itself. Both pathways converge and lead to the formation of the membrane attack complex. Bars over the letter designations of complement components indicate enzymaticajly active forms and dashed lines indicate proteolytic activities of various components (Taken from Abbas et al., 1991)  19  ....20  30  Figure 4.  Mean haematocrit levels (% RBC) in immunized juvenile rainbow trout over three time trials Means (± 1 SE) with different superscripts differ significantly (P < 0.05) by all pairwise multiple comparison .....39  Figure 5.  Mean leucocrit (% WBC) levels in immunized juvenile rainbow trout over three time trials. Means (± 1 SE) with different superscripts differ significantly (P < 0.05) by all pairwise multiple comparison  Figure 6.  Figure 7.  Figure 8.  Figure 9.  Total white blood cell counts (% white blood cells) in immunized juvenile rainbow trout over three time trials Means (± 1 SE) with different superscripts differ significantly (P < 0.05) by all pairwise multiple comparison  40  41  Percentage of head kidney in immunized juvenile rainbow trout over three time trials. Means (+ 1 SE) with different superscripts differ significantly (P < 0.05) by all pairwise multiple comparison  44  Percentage of total kidney in immunized juvenile rainbow trout over three time trials. Means (±1 SE) with different superscripts differ significantly (P < 0.05) by all pairwise multiple comparison  45  Mean lysozyme activity levels in immunized juvenile rainbow trout over three time trials. Means (± 1 SE) with different superscripts differ significantly (P < 0.05) by all pairwise multiple comparison .....48  viii  Figure 10.  Figure 11.  Mean agglutinating antibody titres in immunized juvenile rainbow trout over three time trials. Means (+ 1 SE) with different superscripts differ significantly (P < 0.05) by all pairwise multiple comparison Mean haemolytic activity of plasma in juvenile rainbow trout at three different times following immunization Means (± 1 SE) with different superscripts differ significantly (P < 0.05) by all pairwise multiple comparison  {  ix  49  50  Acknowledgments  I would like to express my gratitude to my supervisor, Dr. George K. Iwama for his faith in my abilities and for his support of the study.  I would also like to express my  appreciation to Microtek Research and Development Ltd. for their assistance in the study, particularly J6zsef Sovenyi for initiating the study and Julian Thornton for helping me complete it. Funding for this research was provided by a Canadian Bacterial Diseases Network operating grant to Dr. Iwama and by a Science Council of British Columbia GREAT award to myself.  I would also like to thank all of the inhabitants of the Fishlab for their guidance, support and friendship through my term as a Masters student. In particular, I want to express my sincere gratitude to: John Morgan for all his help with the respirometer and his patience for my unending problems with it ; Shannon Balfry for her guidance in the immunological aspects of my research and her heart stopping questions ; Ellen Teng for her laughter; and Grace Cho for her boundless emotional support. I would also like to extend a warm appreciation to my wonderful friend Eva Ziduliak, thanks so much for your enthusiastic energy and continuous faith in me. But most importantly, I would like to thank my significant other, Kirk Kohn, without whose ceaseless love, support and patience, I could not have completed this work.  x  General Introduction  The West Coast of British Columbia is an ideal location for the aquaculture industry which is a thriving part of our economy (Little and Keller, 1988 ; Spence et al., 1989). However, a net pen is not always the ideal location for a salmon. The intensive aquaculture practiced on our coast means that the fish are subjected to frequent high levels of stress from handling, extremely high densities and other factors such as low oxygen concentrations and algal blooms in the water. Such stressors may predispose the animals to diseases which may otherwise not be a problem (Peters and Schwarzer, 1985 ; Pickering et al., 1988 ; Maule et al. 1989 ; Schreck et al., 1991 ; Sumpter, 1992).  A widely accepted definition of stress was made by Brett (1958) who stated 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."  Stress can compromise the immune system in fish thereby leading to an increased susceptibility to disease (Anderson, 1990). Where acute stress may actually aid a fish in defending itself against pathogenic invasion by elevating levels of circulating stress hormones such as Cortisol (Maule et al., 1989), the chronic stress involved in culture  generally results in immunosuppression and, subsequently, invasion by a pathogen is more likely to occur (Wedemeyer, 1976).  In addition, stressors can reduce an animal's  physiological performance (Schreck, 1990). Growth, swimming ability and energy usage  1  may be affected by chronic stressors, resulting in slower growth and poor performing animals.  Some of the most problematic bacterial diseases on the west coast include bacterial kidney disease, vibriosis and furunculosis. These diseases can sometimes be treated through the use of antibiotics and chemotherapeutants (Mitchell, 1992 ; Stoffregen et al., 1993 ; Brackett, 1992), although this is generally undesirable for a number of reasons. Microorganisms can develop resistance to the chemotherapeutic agents which are expensive to use and the chemical agents must be cleared from the fish's tissues prior to harvesting.  In addition, there are few chemotherapeutants licensed for use in the  aquaculture industry and it is for these and other reasons that vaccination is a desirable alternative for a culturist. Furunculosis has been an economic problem in fish culture worldwide (Paterson et al., 1988) and in British Columbia the disease is becoming more prevalent as aquaculture is shifting from traditional west coast species of salmon, to production of the Atlantic salmon (Salmo salar) (Sheppard, 1992). Presently, control of the disease in British Columbia is mainly through vaccination and, while these practices have helped to lower mortalities, they have not yet been successful in eliminating of the disease.  Furunculosis  Aeromonas salmonicida,  the etiological agent of furunculosis, is one of the oldest  described pathogens of fish having first been described in brown trout in 1894.  It is an  aerobic Gram-negative, non-motile bacterium which, when grown on trypticase soy agar (TSA), produces a characteristic brown pigment (Michel, 1980). The disease may be found in both salmonids and non-salmonids, farmed and wild, in both salt and fresh water (Evelyn, 2  1971 ; Hastings, 1988). Infection may occur via the gastrointestinal tract, the skin, or through uptake by the gills. The disease is primarily associated with rising and elevated water temperatures of 12°C or greater (Hastings, 1988), but mortalities may occur at temperatures as low as 4-6°C. Outbreaks of the disease may be precipitated by stressors such as handling, grading, overcrowding, transport and transfer; circumstances which are unavoidable in a farm situation.  In an acute epidemic, the first sign of furunculosis is generally an increase in mortalities. In such an outbreak, there may be little change in the external signs of the fish. There may be a slight reddening at the base of the fins. Internally, the kidney and spleen may be swollen and soft.  In a chronic case of furunculosis, there may be a slow increase in mortalities. The fish will generally lose their appetite, and a variety of lesions may form. The skin darkens and the fish become lethargic (Hastings 1988) The lesions are large liquefied regions of muscle necrosis and are typically found in the flank muscles and contain blood tinged fluid. The muscle surrounding the lesions is often red and soft. The lesions are not always present, and in some cases, petechial hemorrhaging will occur on the stomach wall, swim bladder, adipose tissue, gonads and heart. In most typical cases, the spleen and kidney are necrotic (Post 1987). In addition, there may be an inflammation of the lower intestine (Hastings 1988). Fish which survive an infection become carriers of the disease, shedding the bacteria into the water and, consequently, may infect other fish in the vicinity.  Virulent strains of Aeromonas possess an additional layer (A-layer) which surrounds their cell membrane. The A-layer is thought to aid in promoting bacterial uptake. All virulent 3  strains of Aeromonas salmonicida possess this layer but its presence is not necessarily indicative of virulence (Olivier, 1990). Olivier (1990) showed that all virulent forms of A. salmonicida  posses an A-layer, autoaggregate in liquid culture, and were positive on  Coomassie brilliant blue and Congo red media. However, a group of avirulent A+ isolates possessing the same characteristics but without the capability of causing disease were identified. For this reason, in vitro tests cannot predict the virulence of the bacterium and only an in vivo challenge can be used to assess this.  Vaccines  Since 1942, attempts to generate a vaccine against furunculosis have been met with limited success (Duff, 1942). Because the bacterium possesses several potential virulence factors, it has been extremely difficult to formulate an efficient vaccine (Newman and Majnarich, 1985). In addition, it has been shown that immunization by injection provides a dose related protection response against challenge (Newman and Majnarich, 1985).  Live, attenuated bacterins have demonstrated positive results and high potential for use in aquaculture (Cipriano and Starliper, 1982 ; Thornton et al., 1991 ; Thornton et al., 1992) But there is as yet no vaccine that can provide 100% protection to immunized fish. Attempts have been made to passively immunize fish with antisera from rainbow trout and rabbit with some success (Spence et al., 1965 ; Cipriano, 1982 ; Cipriano, 1983 ; McCarthy et al., 1983 ; Olivier et al., 1985). Many cellular products and protein fractions!of A. salmonicida  have been studied in an attempt to use these as vaccines, but none, have  generated a consistent immunity (McCarthy et al., 1983 ; Ellis et al., 1988) Vaccines have been given intramuscularly, intraperitonealy, orally and through bath immersions, with and 4  without boosters in attempts to develop the most effective means of delivering protective immunity to fish (Antipa and Amend, 1977 ; McCarthy et al., 1983 ; Johnson and Amend, 1984 ; Rodgers and Austin, 1984 ; Newman and Majnarich, 1985 ; Nikl et al., 1991 ; Anderson and Jeney, 1992 ; Davidson et al., 1993 ; Jeney and Anderson, 1993 ; Nikl et al., 1993).  Vaccines have been prepared from both virulent and avirulent (Olivier et al., 1985), aggregating and non-aggregating strains (Udey and Fryer, 1978). From all this work, it has been demonstrated that an intraperitoneal injection prepared from whole, killed, virulent cells will result in the most intense immune response (Olivier et al., 1985 ; Davidson et al., 1993) and that there is often a secondary peak in the antibody titre after such an injection (Lamers, 1986 ; Tatner et al., 1987)  The ideal vaccine (Hastings, 1988), should provide protection which is strong, reliable, and prolonged while having the ability to confer additional protection to the fish against atypical strains of the pathogen. In the last decade, the efficacy of vaccines has been further augmented through the use of adjuvants and immunomodulators.  Immunostimulants  Immunostimulants, adjuvants and vaccine carriers are means of enhancing the delivery of and response to, an immunization.  Anderson (1992) defines an  immunostimulant as a chemical, drug or stressor which alters the specific or non-specific immune systems. An adjuvant (e.g. alum) is a substance used to enhance antigenicity when injected with a vaccine. Vaccine carriers are transporters (such as bentonite clay) for 5  vaccines. A description of the immunomodulators used in the present study, and their biological functions follows.  Levamisole: Levamisole is a synthetic drug which functions as a T-cell stimulator (Siwicki et al., 1990 ; Anderson, 1992). It has been shown to elevate leucocyte and neutrophil numbers, enhance neutrophilic phagocytic activity and myeloperoxidase activity, and stimulate leucocyte migration in carp spawners when the levamisole doses are low (Siwicki, 1987). The drug can act alone as a non-specific defense activator by altering levels of lysozyme, or, it may be used as an adjuvant in conjunction with a vaccine where it can enhance the production of antibodies. The mechanisms by which levamisole acts are not well understood, but it appears that it functions as a microbicidal and biostatic agent on cytoplasmic membranes (Amery and Hdrig, 1984).  Oil Emulsion: Oil emulsions act as depots for holding an antigen in the tissues for slow release of bacterin following immunization (Anderson, 1992).  Microencapsulation in an organic polymer: Long term protection against a disease often requires a booster immunization. Immunization initiates the primary specific immune response which involves the production of antibodies specific to the antigen applied. This primary response also involves the production of memory cells which retain the ability to produce these antibodies at an accelerated rate the next time the antigen is encountered. This is known as the secondary immune response and is the basis for booster immunizations. The use of boosters is 6  invaluable to the control of outbreaks of disease, however, in fish culture, the extra handling involved in a booster is undesirable and the use of biodegradable microcapsules is an attractive alternative. Microencapsulation of a bacterin in an organic polymer provides an extended release of the encapsulated immunogen, thereby giving the effectiveness of a vaccine and booster in one injection (Eldridge et al., 1991).  Muramyl dipeptide (MDP): Muramyl dipeptide is the smallest immunogenic portion of the cell wall of mycobacterial species (Anderson, 1992). It has stimulatory effects oh the T-cells and also has positive effects on the cellular immune response.  p-1,3 Glucans: Glucans are long chain polysaccharides which are extracted from the cell walls of yeasts. They are good stimulators of the non-specific defense mechanisms, having an especially pronounced effect on the activity of T-cells and macrophages (Di Luzio et al., 1984 ; Anderson, 1992). Anderson (1992) suggests that because they are products of potential pathogens, the animals may be predisposed to react to them.  Lipopolysaccharide (LPS): LPS is generally accepted as a B-cell stimulator (Anderson, 1992) and it has been demonstrated to have anti-tumor effects and profound effects on the haematopoeitic tissues (Behling and Nowotny, 1979). Fish are predisposed to react to LPS because the lipid-A fraction of the repeating units of polysaccharide-polysaccharide-lipid-A is structurally similar  7  in all Gram-negative species and it is within this fraction that the immunomodulatory activity is thought to occur (Jacobs, 1981).  Objectives of the Study  The objectives of this study were to investigate the effects that various injectable Aeromonas salmonicida vaccines, adjuvanted or otherwise, would have on selected variables of the immune system, the physiology, and the performance of juvenile salmonids. Data were collected on the influence vaccines had on oxygen consumption, and growth of salmonid fish in correlation with some physiological and immunological parameters.  There have been observations both in field and in laboratory settings which indicate that following vaccination against furunculosis, moderate decreases in the growth of immunized fish occurs and that the addition of adjuvants may further decrease the growth rate (Sovenyi, unpublished ; Lillehaug et al., 1992).  It has been speculated that a decrease in growth rate following immunization against furunculosis may be due to an increased usage of nutrients by the fish in order to exert the immune response (Sovenyi, unpublished). The fish may be producing a substantial amount of new immunogenic cells in the haemopoeitic organs which in turn are synthesizing antibodies, complement components and many other substances. Such substances would have to be produced at the expense of other processes and a subsequent decline in growth rate could be explained by a reduced availability of nutrients for anabolic processes. ,  8  Chapter 1. Effects of Adjuvanted Aeromonas salmonicida Vaccines on Growth and Oxygen Consumption Rate of Juvenile Rainbow Trout {Oncorhychus mykiss).  9  Introduction Vaccination by intraperitoneal injection imposes stress on fish due to the handling involved (Lamers, 1986). Such a stressor may cause fish to go off feed for a period of time and to display altered levels of blood hormones (Maule et al., 1989). But after a relatively short time period, these effects diminish (Maule et al., 1989 ; Schreck et al., 1991). With the use of adjuvants in such vaccines, longer lasting effects may be noticed if vaccines themselves prove to have stress effects on the animals.  Growth measurements are frequently used to assess a fish's response to an altered food resource (Rice, 1990), or to a modified environment (Morgan and Iwama, 1991). However, there have been few studies demonstrating effects of vaccination on the growth of fish (Lillehaug, 1992).  Field and laboratory observations seem to indicate that  vaccination against furunculosis does result in decreased growth rates (Sovenyi, unpublished).  Respirometry has been used extensively by many investigators as a means of determining a fish's physiological state in response to its environment by measuring energy and oxygen utilization (e.g. Morgan & Iwama, 1991; Brauner, Shrimpton & Randall, 1992). However, most investigators have studied the effects of the external environment on the fish's ability to perform by examining the effects of salinity changes on a fish's oxygen consumption rate and swimming performance. To date, there have been no concerted studies of this type involving the fish's physiological response to vaccination against diseases.  10  Materials and Methods  Fish Stocks Three batches of domesticated juvenile rainbow trout {Oncorhynchus mykiss) were obtained from Westcreek Trout Farms, Mission, B.C., in March 1993, August 1993, and February 1994.  Fish were maintained at the South Campus Aquaculture Unit of the  University of British Columbia for several weeks to acclimate them to dechlorinated city water. Fish were then transported to the MacMillan Building laboratory and maintained in a 1000 L tank for a week prior to moving them into 16, 80 L tanks in which the experiments were performed.  The experimental design for each trial is shown in Table 1. At the beginning of each trial, fish were randomly distributed among the 16 tanks and allowed to acclimate for one week prior to vaccination. Fish were fed by hand at a rate of 2% of body weight per day using a pelleted, commercial dry feed. Random samples of 10 fish were taken at 4 week intervals throughout the experiments and weights were measured to determine the amount of feed to be supplied to each tank.  Following acclimation, fish were mildly anaesthetized in an aerated tricaine methanesulfonate (MS-222) solution in water (30 mg/L) (Iwama and Ackerman, 1994), measured for length (to the nearest mm) and weight (to the nearest 0.01 g), and immunized by intraperitoneal injection of 0.1 mL of each vaccine preparation. Table 2 outlines the vaccines which were used in the experiments and the dose of adjuvants  11  which were combined with them. Vaccines were prepared by Microtek Research and Development Ltd., in Victoria, B.C. Feed was withheld from fish 24h prior to vaccination to reduce stress on the animals.  Table 1. Experimental conditions for juvenile rainbow trout in three time trials. Time Trial Parameter  First  Second  Third  Timing  May - July 1993  Sept. - Dec. 1993  Apr. - Sept. 1994  Duration  5 weeks  12 weeks  24 weeks  Average Temperature (°C)  11  10  12  Temperature Range (°C)  10-12  14-8  10-17  Photoperiod (h)  12h light, 12h dark (constant)  12h light, 12h dark (constant)  12h light, 12h dark (constant)  Initial number of fish per tank  40  45  40  Initial mean weights (g)  15  12  10  12  Table 2. Vaccine Preparations Treatment group  Immunization  1  0.85% sterile saline  2  Formalin killed Aeromonas salmonicida bacterin  3  A. salmonicida  bacterin with 30 mg/kg Levamisole  4  A. salmonicida  bacterin suspended in emulsified oil  5  A. salmonicida  6  A. salmonicida  7  A. salmonicida  bacterin microencapsulated in the organic polymer poly-(lactide-coglycolide) bacterin microencapsulated with 10% v/v mycobacterial muramyl dipeptide  bacterin microencapsulated with 100% v/v Vita Stim Taito (70% Saccharomyces glucan)  8  cerevisiae  P-1,3  A. salmonicida bacterin microencapsulated with 10% v/v Vibrio anguillarum lipopolysaccharide (LPS)  Respirometry For each trial, respirometry was used to measure oxygen consumption rates halfway through each trial (3, 6 , and 12 weeks). Oxygen consumption measurements were made during exercise as a means of estimating metabolic rates of the test fish. Fish were placed in a modified Brett-type respirometer (Gehrke et al., 1990) which was further modified to hold a total volume of 5.3 L for use with juvenile salmonids.  13  For each experiment, a preliminary sample of unvaccinated fish were run in the respirometer to determine the critical swimming velocity. Critical swimming speed is useful for measuring swimming speeds of individual fish of the same species. Fish with differing swimming abilities may be compared by expressing the swimming speed as Ucm- To measure this, fish are subjected to stepwise increases in swimming velocity until fatigue occurs (Brett, 1964). Fish were forced to swim at a velocity of 1 body length per second (bl/s). Fish were then subjected to hourly 1 bl/s increases in water velocity until the animal fatigued.  LUtS  were then calculated by adding the velocity of the most recently completed  increment and the product of the proportion of the fatigue increment completed (length of time the fish swam at the final velocity divided by 1 h) and the increase in velocity of that increment (1 bl/s) (Brett, 1964 ; Brauner et al., 1992). Fatigue was defined as the point at which a fish could not remove itself from the electrified screen at the posterior of the respirometer chamber. Values was averaged and divided in half, and fish used in the respirometry experiments were swum at this speed (50% LUt), a value roughly equal to 1.5 bl/s.  Water temperature was maintained at the same level as that in the tanks through the submergence of the entire respirometer in a flow through water bath with cooling coils placed in it. The water temperature in the respirometer was maintained at 10°C during all three trials andfishwere all of a similar size and weight.  Because of the large number of fish to be tested and the relatively short duration of the experiments, three fish were run in the chamber each day. Each day threefishwere mildly anaesthetized and their weight and length were measured. These fish were then 14  placed in a three chambered, black perspex flow through chamber and left overnight. The chambers in this box were of roughly the same size as that of the respirometer. The following morning, one fish was removed from the black chamber and placed in the respirometer chamber. This chamber was then wrapped in black plastic to reduce the stress on the fish due to visual disturbance. The entire respirometer was then covered and the water flow was increased to 1.5 bl/s. The fish was allowed 30 min to acclimate to this water flow prior to any measurements being taken. After 30 min, the respirometer was closed off and the decline in water oxygen concentration was measured for 60 - 90 min. At the end of the trial, the fish was removed and returned to the stock population. The same procedure was repeated for the remaining two fish.  Oxygen measurements were taken every 2 min using the LabTech Notebook Data Acquisition system. Because measurements of oxygen consumption can be affected by water temperature, fish size and activity (Brett and Groves, 1979), efforts were made to keep these variables constant through all the experiments. Feed was withheld for 24 h prior to measurements to ensure that energy was not being partitioned to digestion (Brett, 1964). Trials were run over the same time period each day to minimize diurnal variation in metabolic activity (Brett and Zala, 1975).  To adjust for possible bacterial consumption of oxygen within the system, blank runs (i.e. no fish) were made throughout the experiment. No corrections were necessary.  When the water flow valves into and out of the system were closed off, there was always a curvilinear decline in the oxygen concentration for roughly 10-15 min. This was most likely due to pressure changes within the system (Rombough, 1988; Morgan and 15  Iwama, 1991). Oxygen levels then decreased at a constant rate for a further 30-60 min. Oxygen measurements were recorded as partial pressure of oxygen present in the water in the respirometer. The oxygen content of the water in mg/L was calculated using the conversion factors in Colt (1984).  Oxygen consumption rates (mg/kg/h) were then  estimated over the linear portion of the decline through the use of regression analysis.  M0 (mg/kg/h) = slope(mg/L/s) * time(s) * volume of respirometer(L) * weight(kg") 1  2  Growth Experiments At the end of each of the three experiments, length and weight were measured to determine growth rates and condition factor. Standard length rather than fork length was used as a measurement of body length to reduce error due to fin erosion. Standard length was measured as the distance from the snout to the end of the caudal base (Ricker, 1979 ; Cailliet etal., 1986).  Specific growth rates were calculated for each treatment group as In (Wt * WV ) * d" 1  f  * 100, where Wt is the initial mean weight and Wt is the final mean weight of the fish. f  These values are expressed as percent weight per day. Condition factors were calculated using the formula K = (Wt * L" )* 100 (Ricker, 1975). 3  16  1  Statistical Analyses Data are presented as means and standard errors where applicable. Data were subjected to a normality test and, where it passed, a one way analysis of variance was performed. Where data failed the normality test a Kruskal-Wallis one way analysis of variance was performed on ranked data. Where significant differences were detected, all pairwise multiple comparison tests were used to identify significantly different treatment means (P< 0.05). In the case of data generated in the 5 week and 12 week studies which had equal sample sizes, the Student-Newman-Keuls test was used.  The 24 week  experiment had unequal sample sizes and thus, the Dunn's test was utilized (Glantz, 1981). All analyses were performed using the SigmaStat statistical program (Jandel Scientific, San Rafael, California).  17  Results Growth and Condition  Condition factors were high in all treatment groups in all three trials (Table 5.) compared to values obtained by Brett (1964).  However, there were no significant  differences among treatment groups in any of the three time trials.  Growth rates were not significantly different among treatment groups during the 12 week and the 24 week experiments. However, the 5 week experiment showed significant variation among treatment groups (Figure 1). The microencapsulated vaccine formulation which included the addition of Vibrio anguillarum LPS (group 8), displayed the highest growth rate, which was significantly higher than all other treatment groups. The next highest growth rate was observed in the microencapsulated MDP formulation (group 6), and this showed a significantly higher growth rate than the controls and the simple bacterin. However, there were no significant differences among final body weights in any of the trials (Table 6).  Oxygen Consumption Rate  Oxygen consumption rates did not differ significantly at the end 12 and 24 week experiments, but, at the end of the 5 week experiment, the microencapsulated bacterin adjuvanted with V. anguillarum LPS (group 8) displayed a significantly higher metabolic rate than all other treatments except for the levamisole and the oil emulsion immunizations (groups 3 and 4 respectively) (Figure 2) (Table 7).  18  6  7  8  1.4go  1  3  or $ 2 0  12  OS  o _v  i t T3 O  CO  s£ 1.0Group Number  Figure 1. Specific growth rates of immunized juvenile rainbow trout over three time trials. Means (+ 1 SE) with different superscripts differ significantly (P < 0.05) by all pairwise multiple comparison. 19  650 600 CD  O)  550 500  H  a > 450 CD co ro CM  o  400 350 300 H 250 650  ^  600 H  0  =* 550 CD  -§  500  H  co 450 cu  1 CO  400 -I 350 300  H  250 650 D)  600 H  O)  550  —  500 4  $  450 -  llIIIIII 1  2  3  4  5  6  7  8  Group Number Figure 2. Oxygen consumption rates of immunized juvenile rainbow trout over three time trials. Means (+ 1 SE) with different superscripts differ significantly (P < 0.05) by all pairwise multiple comparison. 20  Discussion  Condition factor is a reflection of a fish's nutritional state or well-being (Busacker et al., 1990) and is measured as the relationship or ratio between length and weight of the fish (weight/length) (Goede and Barton, 1990). It also may be roughly interpreted as an index 3  of growth. It has been used in several studies to define stress levels in fish, where a reduction in condition factor is seen in fish that are subjected to one form of stress or another (Barnes et al. 1984). A decline in condition factor may indicate a reduction of the energy stores in the tissues of thefish(Goede and Barton, 1990) or may indicate an altered feeding behavior (Brown et al., 1987) or an increased metabolic rate (Barton and Schreck 1987 ; Schreck, 1990).  Based on this, the lack of significant differences in the condition  factors among any of the treatment groups indicates that none of the treatments had any effect on the nutritional state or well being of the animals.  Growth is a much more sensitive indicator of the health of a fish population than condition factor as it encompasses more of the factors which have acted on a fish in a period of time and sums the results in an easily measurable secondary characteristic. Specific growth rates are the easiest way to measure the growth of fish as they are the measurement of absolute weight gains over a specified period of time (Busacker et al., 1990 ; Austreng et al. 1987). In the present laboratory study, the growth rate data provided a more sensitive indicator of the effects of the vaccines.  The results of these trials 1  demonstrate that the vaccines which were encapsulated and adjuvanted with LPS and MDP had a positive effect on specific growth rates (Table 3). The LPS adjuvant apparently initially gavefisha growth advantage over all other groups. Similarly, the MDP adjuvanted vaccine gave the same advantage to a lesser degree. Since there was only a difference in specific 21  growth rate during the 5 week trial suggests that there was only a short term growth effect and that by the end of 12 weeks, all the groups were growing at the same rate.  Table 3.  Projected final weights of rainbow trout after a two year growth cycle. Values are computed from The University of British Columbia Aquaculture Production Analysis Computer Program, (Iwama and Fidler, 1989) and based on a mean initial weight of 15 g.  Treatment group  Mean specific growth rate  Extrapolated final weights after2ygrowout(q)  Saline  1.16  1573  Bacterin  1.15  1541  levamisole  1.24  1843  Oil adjuvanted  1.26  1915  Microencapsulated  1.26  1915  Muramyl dipeptide  1.30  2065  P-1,3 glucan  1.25  1879  1.41  2518  V. anguillarum LPS  The lack of differences in the final weights in the present study seems to indicate that the differences in specific growth rates were not enough to result in a sizable difference in final body weight among treatment groups. Perhaps if these fish had been maintained until harvest size was attained, differences would have been seen.  While fish were not  maintained to harvest size, the computer production plan created by Iwama and Fidler (1989), based on the growth model created by Iwama and Tautz (1981), provided a simple means of extrapolating the data. The values in Table 3 demonstrate the differences growth rate exerts on the projected final weights offish treated with different vaccine preparations.  22  Lipopolysaccharide is known to induce a fever state in mammals and has been noted to reduce feeding behavior (Plata-Salaman and Borkoski, 1993 ; Wang et al., 1993) and depress blood amino acid concentrations in rats (Garcia-Martinez et al., 1993). However, this depressed feeding behavior recovers over the course of a day (Kent et al., 1992). In a study on immersion vaccines for furunculosis control in rainbow trout, Rodgers (1990) demonstrated that as a side effect of an immersion vaccine containing Aeromonas salmonicida LPS, fish were significantly larger than control fish. LPS has been shown to reduce the food intake of rats by reducing meal frequency but not meal size (Langhans et al., 1990 ; Langhans et al., 1991 ; Langhans et al., 1993), and it has the effect of inhibiting gastric emptying (Langhans et al., 1990). This indicates that the animals are eating fewer meals and that the food is remaining in the stomach for a longer period. For the fish in these experiments, which were fed once daily and not to satiation, there was no apparent reduction in feeding behavior. If the feed was remaining within the stomach for a longer period of time, these fish may have been able to extract nutrients from it in a more efficient manner. Because the vaccine was encapsulated, the release of LPS was prolonged and, therefore, the fish may have been demonstrating an increased feed utilization to counteract a lower blood amino acid level and a reduction in gastric emptying. As the microcapsules were degraded, the levels of LPS would be decreasing and the fish would alter its feed intake accordingly.  Muramyl dipeptide is also a component of bacterial cell walls and has many of the same properties as LPS. It induces fever and has been shown to cause feeding behavior alteration in rats as well (Langhans et al., 1990 ; Langhans et al., 1991). Experiments in rats have shown the feed intake effects of LPS and MDP to be somewhat alike but different enough that there are obviously different mechanisms involved in the responses of the 23  animals. This seems to correlate with the data found in the vaccination experiments on rainbow trout. Muramyl dipeptide had the effect of increasing the specific growth rate of the fish over a short period of time but the effects were not lasting. It seems apparent that the effects of the MDP were however, not as strong as those of the LPS injected group offish.  Oxygen uptake provides the means for a fish to perform the many aerobic physiological functions required for life. Oxygen provides the major means to convert food to energy allowing the fish to swim, grow, feed, reproduce etc. Metabolism is generally understood to be the intracellular process that consumes substrates and produces byproducts in the course of generating chemically stored energy for both anabolic and catabolic activities (Vander et al., 1990). The higher growth rate displayed by the LPS group corresponds to a higher metabolic rate in this same group. This did not hold true for the MDP treatment though.  It may be assumed that a high metabolic rate would be  indicative of a low growth rate, but this is not necessarily correct. Metabolic rate is indirectly measured as oxygen consumption and it must be emphasized that both catabolism as well as anabolism require oxygen. Therefore, it seems that thefishwhich were injected with the LPS were undergoing anabolism at a higher rate than fish injected with the other treatments. The fact that the MDP treatment did not elicit an increased metabolic rate over the other treatment groups could indicate that it had already peaked at an earlier time, or that it rose at some point between 3 and 6 weeks.  All oxygen consumption rates determined were higher than those values found in the literature forfishin fresh water (Brett, 1965 ; Morgan and Iwama, 1991). This is most likely due to the fact that the fish were not given an overnight acclimation period in the swim tunnels for this series of experiments. This would have been very difficult to do given the 24  number of fish to be tested and the short duration of the experiment. Fish swum at the beginning would have been incomparable to the fish swum at the end due to the differences from time of vaccination. However, care was taken that allfishwere treated in a consistent manner.  Also, oxygen consumption rates were not measured for absolute values.  Comparative values were examined to see the effects of different vaccines in comparison with controls and each other. It was interesting to note that when afishwas left overnight to acclimate and the oxygen consumption rate was determined, this value turned out to be only 5-10% lower than that of the same fish left to acclimate for 30 min.  In summary, growth, and metabolic rates of immunized juvenile rainbow trout appear to be only affected for a short period of time following vaccination. Microencapsulated Aeromonas salmonicida bacterin in conjunction with Vibrio anguillarum LPS appears to provide the juvenile fish with a metabolic advantage over other adjuvants.  Muramyl i  dipeptide appears to aid the fish somewhat in regards to growth, but this is somewhat less clear than the obvious effects of LPS. The higher metabolic rate and higher growth rate of the LPS immunized groups could give such vaccinated fish a slight edge over other vaccines, thereby helping these fish in the event of a disease outbreak due to a development advantage.  25  Chapter 2. Some Immunological and Haematological Effects of Adjuvanted Aeromonas salmonicida Vaccines on Juvenile Rainbow Trout (Oncorhychus mykiss).  26  Introduction  Afish'sfirstline of defense against pathogenic invasion is made up of physical barriers such as the skin and mucous layers. The immune responses are influenced by many factors such as stressors, hormonal changes, seasonal effects, drugs and environmental toxicants (Manning and Mughal, 1985). Fish possess both the natural and the acquired immune responses found in higher vertebrates, but there are some significant differences. The immune system of a fish is less sophisticated than that of a mammal (Ellis, 1982); the main haematopoietic tissues in fish are the spleen, the kidney and the headkidney. The antibodies produced by cells in these regions are of a single Ig M-like class. While there are many differences, there are also many similarities with mammalian systems. There are a host of substances involved in the immune response of afishto infection (see Ingram, 1980 and Fletcher, 1981 for reviews) but some of the most important are antibodies, lysozyme and complement proteins. Much like higher vertebrates, fish are also capable of gaining immunological memory in order to mount a secondary response against a pathogen at a later time (Arkoosh and Kaattari, 1991) and of mounting an inflammatory response in reaction to injury or bacterial invasion (Finn and Nielson, 1971).  Temperature plays a large role in the effectiveness of both the humoral and the cellular immune responses (Ainsworth et al., 1991 ; Dexiang and Ainsworth, 1991). At higher temperatures within afish'stolerated physiological range, fish will mount an immune response much quicker and larger than is possible at lower temperatures. When afishis vaccinated at warmer temperatures to initiate the primary response, a normal secondary response can be elicited at lower temperatures.  27  Stress and disease resistance have long been known to be correlated, but the mechanisms behind the relationship are poorly understood. Stressors may modulate physiological systems, which may cause changes in the endocrine system, and which may be manifested as metabolic changes as the animal struggles to maintain homeostasis (Ellis, 1981). Lysozyme activity has been shown to increase in response to a stressful situation (Mock and Peters, 1990) while the haemolytic activity of the blood often decreases in response to a stressor (Fevolden et al., 1992).  Exogenous immunomodulators are often incorporated into vaccines in an attempt to enhance the immune response of the fish. The bacterin or main vaccine is applied in an attempt to stimulate the specific immune system into producing protective antibodies. The application of immunomodulators in conjunction with a bacterin, is to enhance the nonspecific immune system and boost the activity of the antibody producing cells.  Any deleterious environmental factor, either internal or external in relation to the fish, may act as a stressor on the immune system. Many studies have been undertaken by other researchers to examine the effects of one or more vaccines on the production of antibodies and the fish's ability to withstand a disease challenge (Home et al., 1984 ; Nikl et al., 1991 ; Anderson and Jeney, 1992 ; Jeney and Anderson, 1993). However, little work has been done to correlate these findings with the effects on the natural immune system of the animal.  This is the basis for the second chapter of this thesis. Often a vaccine will  produce high antibody titres in a fish, but the fish will display little resistance to a disease challenge (Cipriano, 1982 ; Michel and Faivre, 1982).  It may be possible that such  immunizations are depressing-the non-specific immune system at the same time.  28  The non-specific immune system has a variety of defenses against a bacterial invasion among which is a complex series of enzymatic proteins which make up the complement series. This series of enzymes is present in the serum of fish (Nonaka et al., 1984 ; Ingram, 1987 ; Sakai, 1983), and upon activation, can produce widespread inflammation as well as bacterial lysis. The system may be activated in one or both of two manners, either through activation of the classical pathway which is antibody dependent, or by the activation of the alternative pathway which is antibody independent (Figure 3). Because the alternative pathway may be activated in the absence of antibodies, it helps in the first line of defense against microbial invasion. In contrast to the alternative pathway, the classical pathway must be activated by complexing an antibody to specific surface antigens on phagocytic cells which is the major mechanism in an antibody-mediated immune response. Once activated, the complement system performs a variety of functions, the most commonly studied is the membrane attack complex which causes the lysis of cells. The activity of the classical pathway can be assayed through the use of sensitized heterologous erythrocytes (Legler et al., 1967; Ingram, 1980 ; Ingram, 1987 ; Yano, 1992).  29  Classical Pathway  Antigen-Antibody Complex (IgM) C1  Activated C1 ; (Classical Pathway + C3 convertase) C4 + C2  -• C4b2a-  C3-»C3b A  7 '•  (Classical Pathway C5 convertase) C4b2a3b (Membrane Attack Complex) -> C5b >C5-9 71 7\ 71 71 C6 C7 C8 C9  C5  C3bBb-* C3bBb3b (Alternative Pathway (Alternative Pathway C3 convertase) C5 convertase)  Factor B Factor D Microbial surfaces, polysaccharides Alternative Pathway  Figure 3. Overview of complement activation pathways. The classical pathway is initiated by C1 binding to antigen-antibody complexes, and the alternative pathway is initiated by C3b binding to various activating surfaces, such as microbial cell walls. The C3b involved in alternative pathway initiation may be generated in several ways, including spontaneously, by the classical pathway, or by the alternative pathway itself. Both pathways converge and lead to the formation of the membrane attack complex. Bars over the letter designations of complement components indicate enzymatically active forms and dashed lines indicate proteolytic activities of various components (Taken from Abbas et al., 1991).  30  Materials and Methods Fish Stocks and Vaccines Refer to Materials and Methods, Chapter 1. Blood Collection Ten fish were randomly netted from each tank and placed in an aerated solution containing a lethal dose of MS-222. Fish were removed immediately upon cessation of opercular motion and weights and lengths were measured. Blood was collected with a 1 mL heparinized syringe from the lateral region of the caudal peduncle. One capillary tube was filled with blood for each fish to measure haematocrit. The remaining blood in the syringe was then transferred to a 1.5 mL polypropylene microcentrifuge tube and spun for 5 min at 13,000 X g in a microcentrifuge to separate the plasma from the blood cells. The plasma fraction was then divided between two 0.5 mL polypropylene microcentrifuge tubes and stored at -70°C until used for the agglutination assay. Fish were packed in ice until they were dissected.  Haematocrit Measurement  Heparinized capillary tubes were filled from the syringe of collected blood and centrifuged at 10,000 X g for 5 min in a microhaematocrit centrifuge.  Haematocrits (%  packed red blood cells by volume) were determined by laying the capillary tubes against a Micro-haematocrit tube reader (Lancer ®, Sherwood Medical, St. Louis, MO) and reading the percent values directly (Klontz, 1994). 31  Leucocrit Measurement  Heparinized capillary tubes were filled from the syringe of collected blood and centrifuged at 10,000 X g for 5 min in a microhaematocrit centrifuge. Leucocrits (percent packed leucocytes expressed as a percentage of whole blood volume (Wedemeyer and McLeay, 1981)), were determined by measuring the height of the buffy layer in the capillary tube with Vernier calipers. Values were calculated as a percentage of the total blood volume in the haematocrit tube as follows: height of the buffy layer height of the total blood volume x 100 % (Morgan and Iwama, 1993).  Differential Blood Cell Counts  Two blood smears were prepared for each fish from the blood collected in the syringe and these were stained using a modified Wright-Giemsa stain (Diff-Quick ®, Baxter, Miami, Florida). For each fish, the most uniformly distributed blood smear preparation was chosen from the two slides and differential blood cell counts were made. For each slide examined, ten fields were viewed, working from one end of the slide to the other, and between 50-100 cells were counted and differentiated per field.  Measurement of the Somatic Indices of the Haematopoeitic Organs  Fish were dissected and the spleen, kidney and head kidney were removed and weighed. The kidney and the head kidney were separated at the anterior point where the  32  bulbous head kidney pinches in and narrows to form the kidney. These weights were then expressed as the percent of the total body weight of the fish.  Weighed amounts of kidney tissue samples were then homogenized with four parts (w/v) sodium phosphate buffer (0.06 M, pH 6.0). The homogenate was centrifuged (10,000 X g , 10 min) and the supernatant was stored at -20°C until assayed for lysozyme activity.  Measurement of Kidney Lysozyme Activity  Lysozyme activity was determined using a modified version of the lysoplate, assay described by Lie et al., (1986) and by Ellis (1990). The assay is based on the lysis of Micrococcus lysodeikticus, a bacterium which is particularly susceptible to lysozyme. A suspension of 0.5 mg/mL Micrococcus lysodeikticus (Sigma M3770), 0.02 M NaCl and 0.50% agarose (Sigma A6013) in phosphate buffer (0.06 M, pH 6.0) was prepared. 25 mL of this solution was dispensed onto sterile petri plates (100 x 15 mm) on a level surface and allowed to solidify at room temperature. Ten holes of 3 mm diameter were punched in each plate. 15 ul of test sample was added to each well. Three replicates were run and two fold serial dilutions (ranging from concentrations of 100 - 20,000 u.g/mL) of hen egg white lysozyme in phosphate buffer (0.06M, pH 6.0) were run simultaneously as standards. These standards were run on several plates alongside the test samples to control for differences in incubation temperature and duration. Lysozyme activity of these standards was based on a turbidimetric assay on which a regression analysis was performed. Samples were incubated overnight at room temperature (20°C) and the diameters of the  33  zones of clearance were measured using calipers. The concentration of lysozyme in each sample was then calculated from the following equation:  Y = A + B (log X)  Where Y = the diameter of the clearance zone (mm) A = the Y intercept of the standard regression line B = the slope of the standard regression line X = lysozyme activity (U/g)  Antigen Preparation  A pure culture of an A layer negative A salmonicida strain was obtained from Microtek Research and Development Ltd. (Victoria, B.C.) and was subcultured at 30°C to maintain the absence of the A-layer. This was then streaked onto plates to create a lawn of growth, and cultured at 30°C for 24 h. Bacterial cells were removed from the plates using sterilized microscope slides and washed with phosphate buffered saline (PBS). This suspension was centrifuged at 1600 x g for 25 min. The cell pellet was then resuspended in phosphate buffered saline to which formalin was added (0.3 % final volume) and cells were incubated in the formalin saline on a magnetic stirrer overnight at room temperature. The formalin killed cell suspension was washed three times in PBS by centrifugation and resuspended to an absorbance of 1.0 at 540 nm. 0.01 % Merthiolate was added to this final suspension and the antigen was stored at 4°C until used in the agglutinating antibody titration assay.  34  Measurement of Agglutinating Antibody Titres  Antibody titres were performed using standard microtitre agglutination methods as outlined by Roberson (1990). Each well of a standard 96 well round bottom microtitre plate was filled with 25 u,L of saline. Plasma samples (25 ul) were added to the first well of each series. Using 25 ul microdiluters (Cooke, USA) two-fold serial dilutions were made along each series. The last well did not receive plasma sample and was used as a negative control.  Two fold serial dilutions of rabbit anti -  A. salmonicida  antiserum (Microtek  Research and Development, Victoria, BC) were used as a positive control alongside the test samples. 25 uL of a suspension of A layer negative  A. salmonicida  (at 1.0 optical  density at 540 nm) was added to each well on the 96 well plate. Plates were then covered, swirled gently and incubated at 14°C overnight.  The following day the plates were read to determine the agglutinin titre. Where the samples showed a pellet on the bottom of the wells as in the negative controls, agglutination was considered to be negative. Wells displaying no pellet or a thin film of cells on the bottom of the well were scored as positive for agglutination.  Agglutination titres are  expressed as the reciprocal of the highest dilution displaying agglutination.  Production of Sensitized Rabbit Red Blood Cells  Trout plasma which had been assayed, by Microtek Research and Development Ltd., for agglutination titre of rabbit erythrocytes by a double dilution series, was used to assess the activity of the Complement system through a simple haemolytic assay. Plasma  35  was diluted to the highest agglutination titre and used to sensitize 50% v/v rabbit red blood cells (RaRBC) in PBS solution. A standard curve for complement activity readings for fish sera was provided by Microtek Research and Development Ltd.  Measurement of the Haemolytic Activity of Plasma  A haemolytic assay in agarose was performed according to Weir (1986).  The  method was slightly modified for use with fish plasma. A 2% agarose solution with 0.02% Merthiolate was prepared and kept at 45°C. Pre-cleaned microscope slides were marked on the back and placed on a level surface. 200 |iL of the sensitized RaRBC's was added to a test tube containing 1800 |iL of the agarose solution, vortexed, and quickly poured evenly onto the microscope slide to cover the entire surface equally. The gel slides were allowed to harden in a moist chamber in a refrigerator before cutting wells of 3 mm diameter.  Blood was centrifuged at 10,000 X g for 10 min to separate plasma and blood cells. 10 |iL of plasma was added to each well. Slides were then replaced into the moist chamber and incubated at 15°C overnight. The following day, the zones of clearance were measured using Vernier calipers and haemolytic activity was read off of the standard curve provided by Microtek Research and Development Ltd.  36  Statistical Analyses  Data are presented as means and standard errors where applicable. Data were subjected to a normality test and, where it passed, a one way analysis of variance was performed. Where data failed the normality test a Kruskal-Wallis one way analysis of variance was performed on ranked data. Where significant differences were detected, all pairwise multiple comparison tests were used to identify significantly different treatment means ( P < 0 . 0 5 ) . In the case of data generated in the  5  week and  1 2 week  had equal sample sizes, the Student-Newman-Keuls test was used.  studies which  The 2 4 week  experiment had unequal sample sizes and so, the Dunn's test was utilized (Glantz,  1981).  All analyses were performed using the SigmaStat statistical program (Jandel Scientific, San Rafael, California).  37  Results Haematocrit  Haematocrit values displayed significant differences at the termination of the 5 week trial but these differences disappeared over the longer trials. At the end of 5 weeks, group 8 (LPS) displayed a significantly higher haematocrit level (P < 0.05) than all other groups (Figure 4, Table 8).  Leucocrit  There were no differences in leucocrit during the 5 week time period, but differences appeared over the longer trial trials. After 12 weeks, the LPS treated group (group 8) displayed values which were significantly lower than all other treatments and the P-1,3 glucan (group 7) treatment resulted in a significantly lower leucocrit percentage than the oil emulsion treatment (group 4). At the termination of the 24 week trial leucocrit values of the oil emulsion and the (3-1,3 glucan treatments (groups 4 and 7 respectively) were significantly greater than controls (Figure 5, Table 9).  Differential White Blood Cell Counts  Differential white blood cell counts showed differences in the percentage of total white blood cells among treatment groups at the termination of the 5 week trial period. However, there were no significant differences in the 12 and 24 week time trials. The p-1,3 glucan treatment (group 7) resulted in the highest level of total leucocytes and was  38  Group Number Figure 4.  Mean haematocrit values (% RBC) in immunized juvenile rainbow trout over three time trials. Means (+ 1 SE) with different superscripts differ significantly (P < 0.05) by all pairwise multiple comparison. 39  Figure 5. Mean leucocrit (% WBC) values in immunized juvenile rainbow trout over three time trials. Means (+ 1 SE) with different superscripts differ significantly (P < 0.05) by all pairwise multiple comparison. 40  8 7 -  O  6 -  « 5 0)  ab  >  "13  3 H  0)  2 -  8  _T_  ab  ab  _T_  ab  b JL  JL  1 -  5  8  6  7A  o ^  5  w £  4  >>CM  o ~ Q co  3  CU  2 H 1  H  llllllll 1  2  3  5  4  6  7  8  I  Group Number  Figure 6. Total leucocytes (% white blood cells) in immunized juvenile rainbow trout over three times trials. Means (± 1 SE) with different superscripts differ significantly (P < 0.05) by all pairwise multiple comparison. 41  significantly higher than the levamisole and LPS treatment (groups 3 and 8 respectively). There were also significant differences between the preceding two groups (3 and 8) and the MDP treatment group (6) (Figure 6, Table 9). There were no significant differences in the percentage of monocytes, thrombocytes, lymphocytes or neutrophils among any treatment groups in any of the time trials (Tables 10 and 11).  Somatic Indices of Haematopoeitic Organs  The kidney, head kidney and spleen were dissected out of the body cavity and weighed to determine the percentage of the body weight these organs comprised. The spleen displayed no significant differences among any of the treatment groups in any of the trials (Table 13). However, the head kidney displayed significant differences among groups in all three of the time trials. (These differences are shown in Figure 7 and Table 12). Over the first experiment of 5 week duration, fish in the MDP treatment group (group 6), the |J-1,3 glucan treatment (group 7), and the lipopolysaccharide treatment (group 8) displayed significantly lower proportions of head kidney mass than fish receiving the levamisole adjuvanted vaccine (group 3). At the termination of the 12 week trial,fishtreated with (3-1,3 glucan (group 7) had a significantly lower percentage of head kidney than fish treated with levamisole or MDP (groups 3 and 6). At the termination of the 24 week study, the LPS immunized group (group 8) and the MDP treatment group (group 6) had significantly higher percentages of head kidney material than did the controls (group 1).  There were no significant differences in the percentage of total kidney tissue among any of the treatment groups at the termination of the 5 week experiment. At the 42  termination of the 12 week study however, there were differences among several of the treatment groups. The LPS and the p-1,3 glucan (groups 7 and 8) treatments resulted in significantly lower proportions of total kidney tissue than seen in the saline, bacterin, levamisole and oil emulsion treatment groups (groups 1, 2, 3, and 4 respectively). The percentage of total kidney in the 24 week study followed a pattern somewhat similar to the head kidney with the MDP and LPS vaccines (groups 6 and 8) resulting in significantly higher levels of total kidney tissue than levels in the saline injected controls (group 1). These two treatments also resulted in a higher total kidney tissue percentage than the oil emulsion,  the  microencapsulated bacterin, and the  encapsulated  p-1,3 glucan  immunizations (groups 4, 5, and 7 respectively) (Figure 8, Table 12).  Kidney Lysozyme Activity  Levels of lysozyme activity in the kidney tissue displayed significant differences among groups in all three of the time trials.  At the end of the 5 week trial, the  microencapsulated bacterin (group 5) conferred a significantly higher lysozyme activity level than did the saline, oil emulsion and LPS immunizations (groups 1, 4, and 8, respectively). There were less distinctive differences seen at the termination of the 12 week experiment, but the bacterin in oil emulsion (group 4) and the MDP adjuvanted microencapsulated bacterin (group 6) resulted in significantly higher levels of lysozyme activity than was present in the saline injected group of fish (group 1). Values are shown in Figure 9 and Table 14.  43  0.35 O) CD =S >%  0.30 A  s  XI CD  so  g)  >,LO CD C CO TJ  0.25 A  T3 CO CD X  0.20  0.35 O) CD  0.30 A  £ g •a  ro  0.25 H  TJ CO CD X  0.20  0.35  Figure 7.  Percentage of head kidney in immunized juvenile rainbow trout over three time trials. Means (± 1 SE) with different superscripts differ significantly (P < 0.05) by all pairwise multiple comparison. 44  1.1 JH  1  2  3  4  5  6  7  8  1  2  3  4  5  6  7  8  1-1 ~\  Group Number  Figure 8. Percentage of total kidney in immunized juvenile rainbow trout over three time trials. Means (+ 1 SE) with different superscripts differ significantly (P < 0.05) by all pairwise multiple comparison. 45  At the termination of the 24 week investigation, the LPS immunized fish (group 8) exhibited the highest levels of kidney lysozyme activity, but, due to high variation, the levels were only significant different among this treatment and those of the simple bacterin (group 2) and the bacterin suspended in the oil emulsion (group 4).  Further to this, the oil  adjuvanted bacterin (group 4) and the simple bacterin (group 2) resulted in kidney lysozyme levels which were significantly lower than the saline injected controls (group 1) (Figure 9, Table 14).  Agglutinating Antibody Titres  Fish displayed significant differences among treatment groups in all three trials. At the termination of the 5 week trial, fish treated with V. anguillarum LPS exhibited the highest levels of circulating antibodies, being significantly higher than the levels in the saline injected controls (group 1), the microencapsulated bacterin (group 5) and the formalin killed simple bacterin (group 2). The saline injected control fish (group 1) displayed significantly lower levels of circulating antibodies than all other groups except for the microencapsulated bacterin treated fish (Figure 10, Table 15). In all treatment groups at 5 weeks however, the levels of circulating antibodies were very low.  The 12 week experiment showed group 4 (oil emulsion) as having significantly higher titres than all other groups. There were no significant differences among any of the other groups, but all levels were higher than those at the end of 5 weeks. At the end of 24 weeks, fish immunized with the LPS (group 8) had the highest level of circulating antibody titre, being significantly higher than all other groups. There were differences among the remaining treatment groups though, with the commercial bacterin, the levamisole 46  adjuvanted bacterin, and bacterin in oil emulsion (groups 2, 3, and 4, respectively) having significantly higher levels than the saline injected group (group 1).  Haemolytic Activity of Plasma  There were no significant differences between the plasma haemolytic activity of any of the treatment groups at either 12 or 24 weeks following vaccination. At 5 weeks postimmunization, the haemolytic activity of the plasma was determined to be higher in all treatments which released their contents slowly, that is, the encapsulated vaccines and the bacterin which was been suspended in emulsified oil all displayed higher levels of complement than the non-encapsulated and non-suspended ones, the bacterin which had been adjuvanted with levamisole produced median levels of activity having significantly higher haemolytic activity than the controls and the simple bacterin, and significantly lower activity than the bacterin in oil emulsion, the microencapsulated bacterin ,and the MDP, p1,3 glucan and LPS adjuvanted vaccines.  The plain bacterin produced no significant  increase in levels of haemolytic activity in comparison to the controls (Figure 10, Table 16).  47  J2 6000 0 0 3: m 5000  i= 3000 < N O W  Figure 9.  Mean kidney lysozyme activity levels in immunized juvenile rainbow trout over three time trials. Means (± 1 SE) with different superscripts differ significantly (P < 0.05) by all pairwise multiple comparison. 48  Figure 10. Mean agglutinating antibody titres in immunized juvenile rainbow trout over three time trials. Means (± 1 SE) with different superscripts differ significantly (P < 0.05) by all pairwise multiple comparison. 49  c  18 w  © 16  If 1 CD CO  X  810 -| 6 4 0  ^  0  llllllll 1  2  3  4  5  6  7  8  Group Number  Figure 11. Mean haemolytic activity of plasma in immunized juvenile rainbow trout over three time trials. Means (+ 1 SE) with different superscripts differ significantly (P < 0.05) by all pairwise multiple comparison. 50  Discussion Haematocrit measures the concentration of red blood cells in whole blood (Fange, 1992) and normal values for rainbow trout range from 30 to 50 % (Blaxhall, 1972). Low values are indicative of an anaemic condition, indicating that fish may not be eating or that they may have osmoregulatory problems. Altered haematocrit values can also be indicative of a stressed state (Casillas and Smith, 1977), where values generally increase in a stress situation.  It has been suggested that a slightly hypoxic situation could be created by  muscular exertion and a higher oxygen demand during stress (Casillas and Smith, 1977) and that such a situation can result in an increase in haematocrit levels by as much as 1030%. Immunostimulants rarely affect haematocrit levels (Siwicki and Anderson, 1993) and normal values are widely varied among fish in the same group. With these points in mind, the data gathered at 5 weeks was confusing. While there were no significant differences in haematocrit levels among any treatments at 12 and 24 weeks, there was a significantly higher level in the LPS treated group at 5 weeks. This increase in haematocrit matched the increase in specific growth rate and metabolic rate for these same fish. If they had a higher oxygen demand, it would follow that they required a higher oxygen carrying capacity and would therefore require more red blood cells in the blood stream to carry it to the tissues for the increased anabolic activities. This enhanced ability to carry oxygen may have enhanced tissue building, and as a consequence, fish would have had an enhanced growth rate. The fact that the haematocrit values were slightly higher in each successive experiment was most probably due to the positive correlation that exists between body weight & length, and haematocrit values (Al-Hassan et al., 1993).  51  The volume of white blood cells circulating in a fish's system may reflect an animal's reaction to a host of physiological conditions (McLeay and Gordon, 1977 ; Fange, 1992). The two methods used for examining the white blood cell content gave different results at different times.  The p-1,3 glucan treatment in the 5 week study resulted in a higher  percentage of white blood cells which reflects an increased level of circulating white cells in the blood. This is in agreement with Jorgensen et al., (1993a) who demonstrated that the injection of P-1,3 glucan into the peritoneal cavity of Atlantic salmon resulted in an increased concentration of macrophages, neutrophils and thrombocytes in that region. The leucocrits however, displayed differences only during the two longer trials. Both measurements are used to determine numbers of white blood cells, but where leucocrit is defined as the packed white blood cell volume of total blood and is expressed as a percentage of whole blood, the total white blood cell counts are described as the ratio of white blood cells to red blood cells and expressed as a percentage of total blood cells. Leucocrit is made up roughly of 95% leucocytes and thrombocytes and about 3% erythrocytes and leucocrit values are not correlated to haematocrit values (McLeay and Gordon, 1977).  Leucocrit values in  salmonids typically range from 1-2% (Wedemeyer et al, 1990) and this method of measuring white blood cell volume is suggested as a simple tool for rapidly assessing changes in the numbers of leucocytes and thrombocytes in fish blood. Changes in white blood cell numbers may be highly representative of a physiological response to a toxic substance (Hickey, 1976) and leucocrit levels have been used as a fairly sensitive tool for detecting physiological stress resulting from handling, crowding and temperature changes in addition to Aeromonas salmonicida infections (Wedemeyer et al., 1983). Such infections have been shown to depress leucocrit values significantly (Wedemeyer et al., 1983). It has also been demonstrated that chronic or sub-acute exposure to stressors may result in an  52  increase in leucocrit values and leucocyte numbers (McLeay and Brown, 1974). Wedemeyer et al. (1983) state that leucocrit is only a rough approximation (although correlated) of white blood cell count and the differences seen in the levels of leucocrit but the apparent lack of differences in the total white blood cell percentages during the second and third experiments may be a result of the very subjective nature of the leucocrit test.  Organosomatic indices have been utilized in several stress-related studies.  They  are generally measured as the ratio of the organ weight being studied, to the body weight of the animal. The organs examined in other studies have ranged from the liver (Barton and Schreck, 1987) to the viscera (Jensen 1980), but for the present study, the spleen and kidneys were examined.  The use of organosomatic indices assumes that there is a  proportionate relationship between fish weight and the ratio of a particular organ (Goede and Barton 1990).  Stressed fish may undergo organosomatic changes resulting, for  example, from altered feeding habits or a mobilized immune system. A decline may be due to reduced feed intake or utilization, and an increase could be a result of the production of products to detoxify the blood system or an increase in cell numbers required to mount an immune response.  The anterior or head kidney is the primary site for haematopoeisis in salmonids and the spleen is an accessory organ for this same process. Studies have shown that stress such as handling can affect haematopoeisis in the kidney and the spleen (Peters and Schwarzer 1985 ; Maule and Schreck 1990). In the present study, there were no observed differences in the spleen index but there were some marked differences in kidney values. The fact that the organosomatic index for the head kidneys in the MDP, p-1,3 glucan and  53  LPS treatments were lower at the end of 5 weeks could be due to the fact that changes in tissues take longer than changes in blood parameters. Changes could also be due to the effects of bacterial endotoxins on haematopoietic tissues.  Behling and Nowotny (1979)  demonstrated that the haemopoietic organs often decrease in size and weight following introduction of bacterial endotoxins. This is followed by a hyperplasia of these organs in order to increase the number of white blood cells in the system.  The significant change in the proportion of head kidney tissue between the MDP treatment group and the LPS & p-1,3 glucan treatment groups at the end of the 12 week study, is most likely explained through the manner which MDP affects the immune system. MDP has been demonstrated to activate phagocytes within the head kidney of rainbow trout (Kodama et al. 1993) and to increase the non-specific immune response in rabbits (Chedid et al. 1982). This would indicate that there was an increased production of immunologically important cells and proteins in the head kidney thereby accounting for an increased organosomatic index of this organ.  It has been shown that MDP and LPS both stimulate the production of interleukin-1 which in turn acts as a growth factor for lymphocytes (Allison and Byars, 1986). As the kidney and head kidney are the production sites for lymphocytes, it would be expected that the proportion of these tissues would increase in relation to the controls over the long term. After 24 weeks, it was apparent that the microencapsulated adjuvanted vaccines were exerting a greater effect on these tissues. The LPS and the MDP adjuvants were the only treatments which resulted in significantly higher organosomatic indices of the head kidney tissues than the controls, and the effects far exceeded the effects of levamisole earlier on.  54  The fact that the total kidney organosomatic indices for these two treatments were also significantly higher than most of the other treatments further supports this statement. As stated previously, LPS and MDP are both extracellular products of bacterial cells and as such, have many similar properties and effects (Mulcahy and Quinn, 1986 ; Langhans et al., 1990 ; Langhans et al., 1991).  Lysozymes are enzymes which may occur in many tissues and secretions of living organisms. They play an important bactericidal role in the fight against infection, primarily through lytic actions on the pathogen's cell wall (see Jolles and Jolles, 1984 for a review). A high level of lysozyme may be desirable when culturing fish because it may aid in guarding against infection in situations where there are high densities of fish.  Such  situations are likely to be stressful to the animals and there may be a high bacterial load (Grinde et al., 1988). Lysozyme is said to have a more important role in fish than it does in mammals as the latter have a more sophisticated specific immune system and are therefore, better equipped to fight off an infection. It has been demonstrated that lysozyme plays a very significant role in the disease resistance of rainbow trout (Grinde, 1989) and that in thosefish,kidney lysozyme levels are generally much greater (up to 14 times greater than in Atlantic salmon) than those present in otherfish(Grinde et al., 1988). In addition to this, the levels of lysozyme present in the kidney tissues are many times greater than elsewhere in the body (Lindsay, 1986). This is beneficial to thefishas the kidney acts as a filter, pulling foreign particles from the blood. The removal of organisms from the circulation can cause pathogens to be localized in the kidney tissues, but a high lysozyme level may reduce the chances of disease occurring. There is a huge variation in lysozyme activity between species offish and quite a large variation within species (Fletcher and White, 1976 ; Grinde et al., 1988 ; Muona and Soivio, 1992 ; Holloway et al., 1993). In carp, lysozyme 55  levels have been shown to be highly dependent on age, where older fish have higher levels and fry display the lowest levels of activity (Studnicka et al., 1986).  The fact that the microencapsulated, non-adjuvanted treatment resulted in such a high lysozyme activity at 5 weeks when compared to the p-1,3 glucan and the oil emulsion treatments is a bit confusing. The high level of lysozyme activity present in the microencapsulated simple bacterin treatment group at the termination of the 5 week experiment, may be explained by the long term delivery of the bacterin. While the design of the microcapsules was intended to enhance the antibody response (Eldridge et al., 1991), it also proved effective at enhancing the lysozyme response.  The extended release of  bacterin could have been mimicking an acute Aeromonas salmonicida infection. Moeyner et al. (1993) demonstrated that Atlantic salmon suffering from an acute furunculosis infection, display significantly increased levels of lysozyme and proteolytic enzyme activity while levels of serum proteins are significantly decreased when compared to control fish. This could then, also explain the low levels of antibodies present in the same treatment group at 5 weeks. The low response of the other adjuvanted treatments may indicate that those immunomodulators extend the processing time of a vaccine whether they are incorporated into a microcapsule or into a depot such as the oil emulsion.  The oil adjuvanted treatment failed to provide fish with more than a short term enhancement of the lysozyme levels at the colder temperatures of the second trial which lasted 12 weeks The difference among these and the control fish was reversed at the termination of the 24 week trial such that the controls had a higher lysozyme response than  56  did fish vaccinated with an oil adjuvanted vaccine.  Looking at the levels of lysozyme  through all three trials, it appears that the activity did not change for that treatment.  When examining the data from the 12 and 24 week trials, it seems that the response to the LPS treatment takes longer than that of the MDP, resulting in longer lasting, and higher levels of lysozyme in the kidney tissues. Fletcher and White (1976) demonstrated seasonal changes in serum lysozyme content in plaice with up to a 70% decrease in lysozyme activity in fish during the coldest period of the year.  This may explain the  observed lower levels of lysozyme the present experiment at the 12 week sampling time as the fish were maintained from mid September to early December. While the temperature in this experiment did not drop below 9°C, the fish did experience a significant decline in temperature during the last month of holding and this could explain the lack of difference among treatment values.  The 24 week study showed that, although the commercially available vaccines (groups 2 and 4, bacterin and oil emulsion respectively) gave fish an enhanced antibody response over control fish, the natural immune system appears to have suffered. However, when a simple bacterin was microencapsulated and combined with LPS as an adjuvant, both the specific and the non-specific immune systems were enhanced over a greater time frame.  Antibody titres followed a logical pattern throughout all three trials. As expected, the LPS treatment resulted in a significantly higher level of circulating antibodies than the controls, the formalin killed simple bacterin, and the microencapsulated bacterin. Vibrio anguillarum  and Aeromonas salmonicida both possess LPS. Because of this, the fish 57  would likely be predisposed to react to LPS, and when combined with a furunculosis vaccine, produce a higher level of antibodies to A salmonicida. There were very low levels of circulating antibodies present in the saline-injected fish. This is explained by the knowledge that rainbow trout have a naturally occurring agglutinin titer against Aeromonas salmonicida  (Krantz et al., 1963 ; Cisar and Fryer, 1974) and non-vaccinated fish display  antibodies to both whole cells of A salmonicida and to LPS isolated from the bacterial cell wall (Thuvander et al., 1993). The fact that the microencapsulated bacterin did not result in a titre which was significantly different from the control but was significantly lower than all other treatments is intuitive. The bacterin was not being released as quickly as it was from the oil adjuvanted vaccine. In addition, the other three microencapsulated vaccines, while releasing their bacterin at the same rate, are also releasing a constant dose of immunomodulator at the same time. The simple bacterin preparation would be expected to result in a higher antibody titer at 5 weeks than its microencapsulated counterpart because it has released all of the bacterin into the peritoneal cavity for processing in one dose while the encapsulated vaccine was releasing antigen at a slower, consistent rate.  Antibody titres normally increase significantly between 3 and 8 weeks after vaccination (Erdal and Reitan, 1992) and usually peak at 3 to 6 months (Krantz et al., 1963 ; Cisar and Fryer, 1974 ; Hara and Inoue, 1976) and this was seen to occur in the present trials. All the titres were high at the termination of the 12 week trials, but the oil adjuvanted bacterin treated fish stood out significantly. The sharp increase in antibody titer in this group of fish is seen by other researchers (Allison and Byars, 1986 ; Adams et al., 1988 ; Buonavoglia et al., 1988 ; Midtlyng et al., 1993) who found that oil adjuvanted vaccines displayed rising and significantly higher antibody titres and protection compared to other immunizations and controls especially 3 and 6 months after vaccination. As previously 58  stated, the temperatures during the second trial were quite low and could have resulted in the low titres seen in the other treatment groups. It is interesting to note that fish vaccinated by injection at low temperatures, displayed better protection in challenge experiments performed in other studies, than did those vaccinated at higher temperatures (Lillehaug et al., 1993) and that the mean levels of antibodies were not higher in these same low temperature treatment groups.  At the end of 24 weeks, the oil adjuvanted treatment group showed a decline in levels of circulating antibodies and were not significantly different from any group other than the controls and the LPS adjuvanted encapsulated treatment group. It was quite obvious that the oil adjuvanted vaccine afforded the greatest potential protection at 3 months but not for much longer afterwards. The LPS treatment groups values were progressively higher in each experiment and it is impossible to know if they had peaked prior to 24 weeks or after. It would be interesting to know how long after 24 weeks the antibody titres would remain high in the LPS treatment group. However, it is obvious that the LPS adjuvanted vaccine provided the greatest degree of circulating antibodies for an extended period of time out of any of the treatments. Lipopolysaccharide and MDP have both been used to successfully enhance the vaccine effectiveness of Salmonella vaccines in mice (Bessler et al., 1990) and it has been demonstrated that antibody producing cells in spleen culture increase in number due to the positive effects that LPS exerts on DNA synthesis (Andersson et al., 1972a ; Andersson et al., 1972b). Levamisole is said to have a similar effect (Anderson et al., 1989 ; Siwicki et al., 1990) but, though this could be true when compared to the controls and the encapsulated bacterin in the first experiment, the effect was not visible in the second and third experiments. LPS has been shown to induce antibody production by the B cells. Velji et al. (1992) and Anderson et al. (1993) demonstrated that Vibrio ordalii LPS 59  provided a strong protective immunity to coho salmon, against a V. ordalii infection. Similarly, Bogwald et al. (1992) showed that Atlantic salmon injected with LPS from V. anguillarum gave fish a high degree of protection against the disease.  Antibody titres are not necessarily indicative of the protection of a vaccine or the survival of a fish (Klontz and Anderson, 1970 ; Michel and Faivre, 1982 ; Cipriano, 1983 ; Olivier et al., 1985 ; Hastings and Ellis 1990 ; Thuvander et al., 1993).  It has been  demonstrated that the outcome of a challenge may be independent of the agglutinating antibody titer present in the serum offish (Cipriano, 1982 ; Michel and Faivre, 1982) and that it is the specificity rather than the quantity of antibodies which are produced that is important (Olivier et al., 1985).  Apparently, the antibodies must act in conjunction with  other factors in the fish's immune system in order to combat a pathogen. Only a successful furunculosis challenge could have provided a clear picture of the protectivity of the vaccines in this study.  Activation of the non-specific immune system is important in vaccination protocols in order to give fish a protective first line of defense against pathogenic bacterial invasions. The activity of the complement system is generally assayed using either CH  50  (specific  antibody-dependent complement-mediated activity) which is performed by using sensitized red blood cells, or SH o (non-specific, spontaneous activity) which is performed with 5  erythrocytes which have not been sensitized (Sakai, 1981).  The assessment of  complement activity is a valuable means of detecting the antigen-antibody complex in an immune response. The assay which was run in this study was an attempt to detect any differences among treatments and was not intended to determine absolute values for complement activity. However, the results suggested that encapsulated vaccines may 60  produce significantly higher levels of complement activity than do the commercial vaccines presently on the market.  LPS has been shown to depress the classical pathway, resulting in lower levels of CH activity in the serum (Sakai, 1984), while at the same time enhancing the alternative 50  pathway. This is logical due to the bacterial origin of LPS. Lipopolysaccharide is a bacterial endotoxin (Chedid et al., 1982) and introduction of this substance into a fish would likely be interpreted on an immunological level as a bacterial invasion. Muramyl dipeptide and p-1,3 glucan, also originating from potential pathogenic sources, would most likely have the same effects.  Lipopolysaccharide and MDP have been shown to act in similar manners  (Langhans et al., 1990 ; Langhans et al., 1991). Sakai, (1983) demonstrated that zymosan (a yeast cell wall fraction) elicited a similarly high activation of the alternative complement pathway.  In the present study, it was shown that these three treatments were not  significantly different from one another in their activation of the complement system, and that they did indeed provide a greater response than the controls, the simple bacterin, and the levamisole adjuvanted bacterin. This probably also explains why the oil adjuvanted and microencapsulated simple bacterin resulted in slightly higher (although not significantly different) levels of haemolytic activity than the encapsulated vaccines adjuvanted with cellular products. That is, the oil adjuvanted and the microencapsulated vaccines were both able to deliver a sustained release of bacterin and stimulate the whole complement system rather than simultaneously activating one part of the system while suppressing the other.  Levamisole has been used in many studies as an immunostimulator (Amery and Horig, 1984 ; Nikl et al., 1991) and thus, would be expected to enhance the ability offish to  61  produce antibody-antigen complexes for processing.  It is intuitive that the complement  levels would be higher in this group than in the plain bacterin. It is equally obvious that it would be expected to result in a lower level than the encapsulated bacterin, as the latter treatment is providing a sustained immunization (Eldridge et al., 1991) rather than a single dose of vaccine.  The use of complement in haemolytic assays to examine the response to vaccination protocols could be a valuable addition to the battery of tests that are employed in the assessment of new vaccines. In addition, complement assays can be used to assess nutritional states (Sakai, 1982) and potentially could be used in the ever widening barrage of tests for effects of environmental toxicants. However, a more sensitive assay such as those described by Ingram (1990) and Yano (1992) would be more accurate as they are used to determine absolute levels of haemolytic activity rather than relative values.  There was a wide range of effects on the variables examined, but it was seen that microencapsulated vaccines exerted a highly favorable impact on the haematology and immunology of juvenile salmonids. LPS had a greater positive impact on these variables in immunized trout than did any of the other treatments. The high short term haematocrit level suggests a better oxygen carrying capability. The high lysozyme levels over the long term are non-specifically protective, reducing the chances of disease.  Antibody titres were  higher in the LPS treated group in each trial, demonstrating that this vaccine held not only the ability to cause high levels of circulating antibodies, but acted to keep them elevated for an extended period. While the bacterin in oil emulsion provided high levels of antibody at 12 weeks and higher than control levels of haemolytic activity at 5 weeks, it failed to demonstrate any long term effects on these variables. 62  A good vaccine must provide  protection quickly and have long lasting defensive effects. Vibrio LPS incorporated into encapsulated injectable vaccine appears to have these qualities.  63  Concluding Remarks  This study examined a number of immunomodulators, and in most cases, they all proved to be superior in the enhancement of the animals immune response and physiological processes in comparison to the use of simple bacterin based vaccines. This work indicates that there is potential for the use of immunomodulators in commercial furunculosis vaccines.  Further, this study has demonstrated the value of the use of  microencapsulation technology for the aquaculture industry. The extended delivery of both vaccine and immunomodulator holds great promise for an effective furunculosis vaccine which maintains long term protection both at the specific and the non-specific levels, while not having a high metabolic cost to the animals in the long term.  The highest level and broadest range of positive responses of all the immunomodulators tested was provided by LPS. The metabolic and growth studies indicated that the physiological effects of furunculosis vaccines are relatively short lived, but that those short term effects may have very important long term impacts. The high oxygen consumption rate and high haematocrit in the LPS treatment group suggests that these fish were afforded an enhanced oxygen carrying capacity in the blood and a heightened rate of anabolic activity. When the computerized production model was run, the higher growth rate of the LPS treated group translated to a phenomenal potential difference in size at harvest for these fish. This would translate into a considerable profit to a culturist. LPS also provided fish with an extremely long period of potential protection through antibody production, while offering non-specific protection in the form of high lysozyme levels. Of all the vaccine formulations tested, Vibrio anguillarum LPS appears to hold the most promise for immunomodulatory use in an encapsulated vaccine. 64  While the oil adjuvant provided by Bio-Med Inc. (Bellvue, WA) induced high levels of antibodies in the system, it was a difficult vaccine to work with. It is very thick and dense and it required warming up before it could be injected in any practiced manner.  Morris et al. (1994) state that microencapsulation technology has the potential to enhance the immune response through both oral and parental routes of immunization. The literature shows that parental routes of immunization show far more success in vaccination protocols. While oral administration shows promise for use in animals where feed intake can be more easily determined (Moldoveanu et al., 1993 ; Ray et al., 1993), injectable vaccine preparations appear more suited to the aquaculture industry.  Glucans have been touted as the miracle immunomodulator for the fish industry (Moore-Clark pamphlet, 1993). They have been shown to provide animals with high levels of protective immunity in comparison to control groups and when compared to selected treatments (Yano et al., 1989 ; Nikl et al., 1991 ; Chen and Ainsworth, 1992 ; Matsuyama et al., 1992). The present study indicated that compared to controls, on the basis of antibody titres at 5 weeks, [3-1,3 glucans could provide fish with an enhanced immune response. However, this was not different from any other treatments except for the simple microencapsulated bacterin. Furthermore, this difference from the control group was not maintained in the following trials of longer duration. Jorgensen et al., (1993b) demonstrated that injection of glucans into the peritoneal cavity, resulted in an enhanced serum lysozyme activity throughout their 3 week experiment. In the present study, the injection of glucans with a bacterin did not afford fish any special protection in the form of a heightened  65  lysozyme activity. In fact, the only variable examined in which p-1,3 glucans demonstrated any superiority over any of the other immunomodulators was in the number of circulating leucocytes.  There are many parts of this investigation which demand further study. The effects of these vaccines over time would have been much clearer had the same fish been maintained over a year long period. If a larger population of fish had been maintained for a full year, more frequent sampling would have been possible and a clearer picture of the development of immune responses and metabolic and physiological changes could have been constructed. However, this was not feasible in the facilities I had for this study.  Disease challenge data would have been valuable to support the predicted protectivity of the vaccines tested. Challenges with Aeromonas salmonicida are difficult due to the nature of the bacteria, and they are often either irreproducible or do not follow a natural route of infection (Michel, 1982). While a bath challenge (McCarthy, 1983 ; Adams et al., 1987) was attempted in this study, results were disappointing. Hirst and Ellis (1994) recently used a 24h bath challenge successfully and this appears to be a more effective means of inducing reproducible challenge results. Cohabitation challenge (Aakre et al., 1994) relies on fish-to-fish transmission of the pathogen, emulating a more natural mode of infection. In the present study, the water temperatures were not conducive to infection in two of the trials and mortalities were almost non-existent in the third. It would be valuable to perform the experiments in a temperature controlled system which was not available in this study. It would also be useful to repeat the study on a species of fish which does not have the naturally high resistance to Aeromonas salmonicida that rainbow trout display. Rainbow  66  trout were used in this study as a model for the more valuable commercial food fish, the Atlantic salmon  (Salmo salar).  It would be interesting to apply the study to these fish and  see how they fare in comparison.  67  Table 4.  Summarized significant results among seven immunization treatments, over three time trials.  Specific growth rate  Metabolic rate  Haematocrit Leucocrit  Total leucocytes  Somatic index of head kidney Somatic index of total kidney  Lysozyme Activity  Antibody titre  Haemolytic activity  5 weeks -LPS greater than all other treatments -MDP greater than controls and bacterin -LPS greater than controls, bacterin, microencapsulated bacterin, MDP and glucan -LPS greater than all other treatments -No differences  -p-1,3 glucan and MDP greater than LPS and levamisole -Levamisole greater than MDP, (3-1,3 glucan, and LPS -No differences  12 weeks -No differences  24 weeks -No differences  -No differences  -No differences  -No differences  -No differences  -LPS lower than all other treatments -Oil emulsion greater than p-1,3 glucan and LPS -No differences  -p-1,3 glucan and oil emulsion greater than controls  -p-1,3 glucan lower than MDP and levamisole -LPS and p-1,3 glucan lower than controls, bacterin, levamisole and oil emulsion  -LPS and MDP greater than controls  -Microencapsulated bacterin greater than controls, oil emulsion, and, LPS -LPS greater than all other treatments -Controls lower than all other treatments  -Oil emulsion and MDP greater than controls  -Controls and bacterin lower than all other treatments -Levamisole lower than oil emulsion, microencapsulated bacterin, MDP, p-1,3 glucan, and LPS  -No differences  -Oil emulsion greater than all other treatments  68  -No differences  -LPS and MDP greater than controls, bacterin, levamisole, oil emulsion, microencapsulated bacterin, and p-1,3 glucan -LPS and controls greater than bacterin and oil emulsion -LPS greater than all other treatments -Controls lower than bacterin, levamisole, and oil emulsion -No differences  References Aakre, R., H.I. Wergeland, P.M. Aasjord, and C. Endressen. 1994. Enhanced antibody response in Atlantic salmon (Salmo salar L.) to Aeromonas salmonicida cell wall antigens using a bacterin containing (3-1,3-M-glucan as adjuvant. Fish Shellfish Immunol. 4: 47-61. Abbas, A.K., A.H. Lichtman, and J.S. Pober. 1991. Cellular and Molecular Immunology. W.B. Saunders Company, Harcourt Brace Jovanovich, Inc., Philadelphia, Pennsylvania. 417 p. Adams, A., N. Auchinachie, A. Bundy, M.F. Tatner, and M.T. Home. 1988. The potency of adjuvanted vaccines in rainbow trout (Salmo gairdneri , Richardson.) and bath vaccines in Atlantic salmon (Salmo salar L.) against furunculosis. Aquaculture. 69: 15-26. Adams, A., W. Leschen, A. Wilson, and M.T. Home. 1987. A bath challenge model for furunculosis in rainbow trout, Salmo gairdneri Richardson, and Atlantic salmon, Salmo salar L. J. Fish. Dis. 10: 495-504. Ainsworth, A.J., C. Dexiang, P.R. Waterstrat, and T. Greenway. 1991. Effect of temperature on the immune system of channel catfish (Ictalurus punctatus) - I. Leucocyte distribution and phagocyte function in the anterior kidney at 10 °C. Comp. Biochem. Physiol. 100A: 907-912. Al-Hassan, L.A.J., H.K. Ahmed, and S.A. Majeed. 1993. Some haematological parameters in relation to the biology of the fish Acanthopagrus latus. J. Environ. Sci. Health, Part A: Environ. Sci. Eng. A28: 1599-1611. Allison, A.C. and N.E. Byars. 1986. An adjuvant formulation that selectively elicits the formation of antibodies of protective isotypes and of cell-mediated immunity. J. Immunol. Methods 95: 157-168. Amery, W.K., and C. Horig. 1984. Levamisole. p. 383-400 In R.L. Fenichel, and M.A. Chirigos (eds.). Immune modulation agents and their mechanisms. Marcel Dekker, Inc. New York, New York. Anderson, C C , R. Rahimpour, and N.R. StC Sinclair. 1993. Mutual antagonism between antigen- and lipopolysaccharide induced antibody production. Immunol. Invest. 22: 531-540. Anderson, D.P. 1990. Immunological indicators: Effects of environmental stress on immune protection and disease outbreaks. American Fisheries Society Symposium 8: 38-50. Anderson, D.P. 1992. Immunostimulants, adjuvants, and vaccine carriers in fish: applications to aquaculture. Ann. Rev. Fish Dis. 2: 281-307.  69  Anderson, DP., and G. Jeney. 1992. Immunostimulants added to injected Aeromonas salmonicida bacterin enhance the defense mechanisms and protection in rainbow trout {Oncorhynchusmykiss). Vet. Immunol. Immunopathol. 34: 379-389. Anderson, DP., G. Jeney, M. Carrilo, L. Dahle, J. Morales, P. Sorgeloos, N. Svennevig, and J. Wyban (eds.). 1993. Immunostimulation and protection from diseases in rainbow trout by glucans given by injection of bath. From Discovery to Commercialization. Spec. Publ. Eur. Aquacult. Soc. 19: 199 Summary only. Anderson, DP., A.K. Siwicki, O.W. Dixon, and E.F. Lizzio. 1989. Immunostimulation by levamisole in rainbow trout {Salmo gairdneri) in vivo. p. 469-478 In W. Ahne, and E. Kurstak (eds.). Viruses of lower vertebrates. Springer-Verlag Pub., Berlin, New York. Andersson, J., G. Moller, and O. SjOberg. 1972a. Selective induction of DNA synthesis in T and B lymphocytes. Cell. Immunol. 4: 381-393. Andersson, J., 0. Sjoberg, and G. Moller. 1972b. Induction of immunoglobulin and antibody synthesis in vitroby lipopolysaccharides. Eur. J. Immunol. 2: 349-353. Antipa, R., and D.F. Amend. 1977. Immunization of Pacific salmon: Comparison of intraperitoneal injection and hyperosmotic infiltration of Vibrio anguillarum and Aeromonas salmonicida bacterins. J. Fish. Res. Board. Can. 34: 203-208. Arkoosh, M.R., and S.L. Kaattari. 1991. Development of immunological memory in rainbow trout {Oncorhynchus mykiss). I. An immunochemical and cellular analysis of the B cell response. Devel. Comp. Immunol. 15: 279-293. Austreng, E., T. Storebakken, and T. Aasgaard. 1987. Growth estimates for cultured Atlantic salmon and rainbow trout. Aquaculture. 60: 157-160. Barnes, M.A., G. Power, and R.G.H. Downer. 1984. Stress related changes in lake whitefish {Coregonus clupeaformis) associated with a hydroelectric control structure. Can. J. Fish. Aquat. Sci. 41: 1528-1533. Barton, B.A., and C.B. Schreck. 1987. Metabolic cost of acute physical stress in juvenile steelhead. Trans. Amer. Fish. Soc. 116: 257-263. Behling, U.H., and A. Nowotny. 1979. Immunostimulation by LPS and its derivatives, p. 165-179 InH. Friedman, T.W. Klein, and A. Szentivanyi. (eds.). Immunomodulation by bacteria and their products. Plenum Press, New York, New York. Bessler, W.G., B. Kleine, L. Biesert, S. Schlecht, R. Schaude, K.H. Wiesmuller, J. Metzger, and G. Jung. 1990. Bacterial surface components as immunomodulators. p. 37-48 In K.N. Masihi, and W. Lange (eds.). Immunotherapeutic prospects of infectious diseases. Springer-Verlag Pub. Berlin, Heidelberg. Blaxhall, P.C. 1972. The haematological assessment of the health of freshwater fish. J. Fish Biol. 4: 593-604.  70  Bogwald, J., K. Stensvag, J. Hoffman, K.O. Holm, and T.O. Jorgensen. 1992. Vaccination of Atlantic salmon, Salmo salar L, with particulate lipopolysaccharide antigens from Vibrio salmonicida and Vibrio anguillarum. Fish Shellfish Immunol. 2: 251-261. Brackett, J. 1992. The regulatory control of furunculosis: Disease and drug regulations. Bull. Aquacult. Assoc. Can. 92: 58-60. Brauner, C.J., J.M. Shrimpton, and D.J. Randall. 1992. Effects of short-duration seawater exposure on plasma ion concentrations and swimming performance of coho salmon (Oncorhynchus kisutcti) parr. Can. J. Fish. Aquat. Sci. 49: 2399-2405. Brett, J.R. 1958. Implications and assessments of environmental stress, p. 69-83 In P.A. Larkin (ed.). Investigations of fish-power problems. H.R. MacMillan Lectures in Fisheries, University of British Columbia. Brett, J.R. 1964. The respirator metabolism and swimming performance of young sockeye salmon. J. Fish. Res. Board Can. 21: 1183-1226. Brett, J.R. 1965. The relation of size to rate of oxygen consumption and sustained swimming speed of sockeye salmon (Oncorhychus nerka). J. Fish. Res. Board Can. 22: 1491-1501. Brett, J.R., and T.D.D. Groves. 1979. Physiological energetics, p. 279-352 In W.S. Hoar, D.J. Randall, and J.R. Brett, (eds.). Fish physiology, Volume VIII. Academic Press, New York, New York. Brett, J.R., and CA. Zala. 1975. Daily pattern of nitrogen excretion and oxygen consumption of sockeye salmon (Oncorhynchus nerka) under controlled conditions. J. Fish. Res. Board Can. 32: 2479-2486. Buonavoglia, C , L. di Trani, F. Gramenzi, R. Zoletto, R, Lelli, and M. Scacchia. 1988. Duration of immunity in swine inoculated with a mono-valent foot and-mouth disease oil emulsion vaccine. J. Vet. Med. 35: 397-401. Busacker, G.P., I.R. Adelman, and E.M. Goolish. 1990. Growth, p. 363-387 InCB. Schreck and P.B. Moyle (eds.). Methods for fish biology. American Fisheries Society, Bethseda, Maryland. Cailliet, G.M. 1986. Fishes: a field and laboratory manual on their structure, identification, and natural history. Wadsworth Pub. Belmont, CA. 194 p. Casillas, E., and L.S. Smith. 1977. Effect of stress on blood coagulation and haematology in rainbow trout (Salmo gairdneri) J. Fish. Biol. 10: 481-491. Chedid, L.A., M.A. Parant, F.M. Audibert, G.V. Riveau, F.P. Parant, E. Lederer, J.P. Choay, and P.L. Lefranccier. 1982. Biological activity of a new synthetic muramyl dipeptide adjuvant devoid of pyrogenicity. Infec. Immun. 35: 417-424.  71  Chen, D., and A.J. Ainsworth. 1992. Glucan administration potentiates immune defence mechanisms of channel catfish, Icta/urus puctatusRafinesque. J. Fish. Dis. 15: 295304. Cipriano, R.C. 1982. Immunization of brook trout (Salvelinus fontinalus) against Aeromonas salmonicida: Immunogenicity of virulent and avirulent isolates and protective ability of different antigens. Can. J. Fish. Aquat. Sci. 39: 218-221. Cipriano, R.C. 1983. Resistance of salmonids to Aeromonas salmonicida. Relation between agglutinins and neutralizing activities. Trans. Amer. Fish. Soc. 112: 95-99. Cipriano, R.C, and C E . Starliper. 1982. Immersion and injection vaccination of salmonids against furunculosis with an avirulent strain of Aeromonas salmonicida. Prog. FishCult. 44: 167-169. Cisar, J.O., and J.L. Fryer. 1974. Characterization of &c\\\-Aeromonas salmonicida antibodies from coho salmon. Infec. Immunity 9: 236-243. Colt, J. 1984. Computation of dissolved gas concentrations in water as functions of temperature, salinity, and pressure. American Fisheries Society Special Publication no. 14 154 p. Davidson, G.A., A.E. Ellis, and C J . Secombes. 1993. Route of immunization influences the generation of antibody secreting cells in the gut of rainbow trout (Oncorhynchus mykiss) Devel. Comp Immunol. 17: 373-376. Dexiang, C , and J. Ainsworth. 1991. Effect of temperature on the immune system of channel catfish (Ictalurus punctatus) - II. Adaptation of anterior kidney phagocytes to at 10°C Comp. Biochem. Physiol. 100A: 913-918. Di Luzio, N.R., R.B. McNamee, E.L. Jones, D.L. Williams, R.L. Fenichel, and M.A. Chirigos (eds.). 1984. Influence of host tumor relationships on response to the biological response modifiers, glucan, and methyl palmitate. Immunol. Ser. 25: 437-445. Duff, D.C.B. 1942. The oral immunization of trout against Aeromonas salmonicida. Immunol. 44: 87-94.  J.  Eldridge, J.H., J.K. Staas, J.A. Meulbroek, J.R. McGhee, T.R. Tice, and R.M. Gilley. 1991. Biodegradable microspheres as a vaccine delivery system. Molec. Immunol. 28: 287-294. Ellis, A.E. 1981. Stress and the modulation of disease, p. 147-170 In A.D. Pickering (ed.). Stress and fish. Academic Press inc., London. Ellis, A.E. 1982. Differences between the immune mechanisms of fish and higher vertebrates, p. 1-30 In R.J. Roberts (ed.). Microbial diseases of fish. Academic Press Inc., London.  72  Ellis, A.E. 1990. Lysozyme assays, p. 101-103 In J.S. Stolen, T.C. Fletcher, DP. Anderson, B.S. Roberson, W.B. van Muiswinkel (eds.). Techniques in fish immunology. SOS Publications, Fair Haven, New Jersey. Ellis, A.E., A.S. Burrows, T.S. Hastings and K.J. Stapelton. 1988. Identification of Aeromonas salmonicida extracellular protease as a protective antigen against furunculosis by passive immunization. Aquaculture 70: 207-218. Erdal, J.I., and L.J. Reitan. 1992. Immune response and protective immunity after vaccination of Atlantic salmon (Salmo salar L.) against furunculosis. Fish Shellfish Immunol. 2: 99-108. Evelyn, T.P.T. 1971. An aberrant strain of the bacterial fish pathogen Aeromonas salmonicida isolated from a marine host, the sablefish (Anoplopoma fimbria), and from two species of cultured Pacific salmon. J. Fish. Res. Board Can. 28: 16291634. Fange, R. 1992. Fish red blood cells, p. 1-54 In W. Hoar, and D.J. Randall (eds.). Fish physiology vol. XIIB. Academic Press Inc., London. Fevolden, S.E., T. Refstie, and K.H. Reed. 1992. Disease resistance in rainbow trout (Oncorhynchus mykiss) selected for stress response. Aquaculture. 104: 19-29. Finn, J.P., and N.O. Nielson. 1971. The inflammatory response of rainbow trout. J. Fish Biol. 3: 436-478. Fletcher, T.C. 1981. Non-antibody molecules and the defence mechanisms offish. In A.D. Pickering (ed.). Stress and Fish. Academic Press Inc., London. Fletcher, T.C, and A. White. 1976. The lysozyme of the plaice Pleuronectes platessa L. Comp. Biochem. Physiol. 55B: 207-210. Garcia-Martinez, O, M. Llovera, F.J. Lopez-Soriano, and J.M. Argiles. 1993. The effects of endotoxin administration on blood amino acid concentrations: similarities with sepsis. Cell. Mol. Biol. 39: 537-542. Gehrke, P.C., L.E. Fidler, D.C. Mense, and D.J. Randall. 1990. A respirometer with controlled water quality and computerized data acquisition for experiments with swimming fish. Fish Physiol. Biochem. 8: 61-67. Glantz, S.A. 1981. Primer of Bio-Statistics 3rd ed. McGraw-Hill, Inc., New York, New York. 440 p. Goede, R.W., and B.A. Barton. 1990. Organosomatic indices and an autopsy-based assessment as indicators of health and condition of fish. American Fisheries Society Symposium 8: 93-108. Grinde, B. 1989. Lysozyme from rainbow trout, Salmo gairdneri Richardson, as an antibacterial agent against fish pathogens. J. Fish. Dis. 12: 95-104. 73  Grinde, B., 0. Lie, T. Poppe and R. Salte. 1988. Species and individual variation in lysozyme activity in fish of interest in aquaculture. Aquaculture. 68: 299-304. Hara, T., and K. Inoue. 1976. Vaccination trials for control of furunculosis of salmonids in Japan. Fish. Path. 10: 227-235. Hastings, T.S. 1988. Furunculosis Vaccines, p. 93-111 In A.E. Ellis (ed.). Fish vaccination. Academic Press Inc., London. Hastings, T.S. and A.E. Ellis. 1990. Detection of antibodies induced in rainbow trout by different Aeromonas salmonicida vaccine preparations. J. Aquat. Anim. Health. 2: 135-140. Hickey, CR. Jr. 1976. Fish haematology, its uses and significance. New York Fish Game J. 23: 170-175. Hirst, I.D., and A.E. Ellis. 1994. Iron-regulated outer membrane proteins of Aeromonas salmonicida are important protective antigens in Atlantic salmon against furunculosis. Fish Shellfish Immunol. 4: 29-45. Holloway, H.L. Jr., CA. Shoemaker, and CA. Ottinger. 1993. Serum lysozyme levels in paddlefish and walleye. J. Aquat. Anim. Health. 5: 324-326. Home, M.T., R.J. Roberts, and M. Tatner. 1984. The effects of the use of potassium alum adjuvant in vaccines against vibriosis in rainbow trout, Salmo gairdneri Richardson. J. Fish Dis. 7: 91-99. Ingram, G.A. 1987. Haemolytic activity in the serum of brown trout, Salmo trutta. J. Fish Biol. 31 (Supplement A): 9-17. Ingram, G.A. 1980. Substances involved in the natural resistance of fish to infection - a review. J. Fish Biol. 16: 23-60. Ingram, G.A. 1990. Complement-fixation test. p. 25-44 In J.S. Stolen, T.C Fletcher, D.P. Anderson, B.S. Roberson, W.B. van Muiswinkel (eds.). Techniques in fish immunology. SOS Publications, Fair Haven, New Jersey. Iwama, G.K., and P.A. Ackerman. 1994. Anaesthetics, p. 1-15 InP.W. Hochachka, and T.P. Mommsen (eds.). Biochemistry and molecular biology pf fishes, vol. 3. Elsevier Science B.V., Amsterdam. Iwama, G.K., and L.E. Fidler. 1989. The University of British Columbia Aquaculture Analysis Program. A Research and Development Project Funded by the British Columbia Ministry of Agriculture and Fisheries. Iwama, G.K., and A.F. Tautz. 1981. A simple growth model for salmonids in hatcheries. Can. J. Fish. Aquat. Sci. 38: 649-656. Jacobs, D.M. 1981. Immunomodulatory effects of bacterial LPS. J. Immunopharm. 3: 119132. 74  Jeney, G., and DP. Anderson. 1993. Enhanced immune response and protection in rainbow trout to Aeromonas salmonicida bacterin following prior immersion in immunostimulants. Fish. Shellfish Immunol. 3: 51-58. Jensen, A.J. 1980. The 'gut-index', a new parameter to measure the gross nutritional state of Arctic char, Salvelinus alpinus (L.) and brown trout, Salmo trutta L. J. Fish Biol. 17: 741-747. Johnson, K.A. and D.F. Amend. 1984. Potential for immersion vaccination against A. salmonicida. J. Fish. Dis. 7: 101-105. Jolles. P, and J. Jolles. 1984. What's new in lysozyme research? Always a model system, today as yesterday. Molec. Cell. Biochem. 63: 165-189. Jorgensen, J.B., H. Lunde, and B. Robertsen. 1993a. Peritoneal and head kidney cell response to intraperitonealy injected yeast glucan in Atlantic salmon, Salmo sa/arL. J. Fish Dis. 16: 313-325. Jorgensen, J.B., G.J.E. Sharp, C.J. Secombes, and B. Robertsen. 1993b. Effect of a yeastcell-wall glucan on the bactericidal activity of rainbow trout macrophages. Fish Shellfish Immunol. 3: 267-277. Kent, S., K.W. Kelley, and R. Dantzer. 1982. Effects of lipopolysaccharide on foodmotivated behavior in the rat are not blocked by an interleukin-1 receptor antagonist. Neurosci. Lett. 145: 83-86. Klontz, G.W. 1994. Fish Hematology, p. 121-131 In J.S. Stolen, T.C. Fletcher, A.F. Rowley, J.T. Zelikoff, S.L. Kaattari, S.A. Smith (eds.). Techniques in fish immunology - 3. SOS Publications, Fair Haven, NJ. Klontz, G.W., and Anderson. 1970. Oral immunization of salmonids: a review, p. 16-20. In S.F. Snieszko (ed.). Symposium on diseases of fishes and shellfishes. Special Publication no. 5 American Fisheries Society, Washington, DC. Kodama, H., Y. Hirota, M. Mukamoto, T. Baba, and I. Azuma. 1993. Activation of rainbow trout (Oncorhynchus mykiss) phagocytes by muramyl dipeptide. Dev. Comp. Immunol. 17: 129-140. Krantz, G.E., J.M. Reddecliff, and OE. Heist. 1963. Development of antibodies against Aeromonas salmonicida in trout. J. Immunol. 91: 757-760. Lamers, C.H.J. 1986. Histopathology of a primary immune response against Aeromonas salmonicida in carp (Cyprinus carpio L.). J. Exp. Zool. 238: 71-80. Langhans, W., G. Balkowski, and D. Savoldelli. 1991. Differential feeding responses to bacterial lipopolysaccharide and muramyl dipeptide. Am. J. Physiol. 261: R650R664.  75  Langhans, W., R. Harlacher, G. Balkowski, and E. Scharrer. 1990. Comparison of the effects of bacterial lipopolysaccharide and muramyl dipeptide on food intake. Physiol. Behav. 47: 805-813. Langhans, W., D. Savoldelli, and S. Weingarten. 1993. Comparison of the feeding response to bacterial lipopolysaccharide and interleukin-1 beta. Physiol. Behav. 53: 643-649. Legler, D.W., E.E. Evans., and H.K. Dupree. 1967. Comparative immunology: serum complement of freshwater fishes. Trans. Amer. Fish. Soc. 96: 237-242. Lie, O., M. Syed, and H. Solbu. 1986. Improved agar plate assays of bovine lysozyme and haemolytic complement activity. Acta. Vet. Scand. 27: 23-32 Lillehaug, A., T. Lunder, and T.T. Poppe. 1992. Field testing of adjuvanted furunculosis vaccines in Atlantic salmon, Salmo salarL. J. Fish Dis. 15: 485-496. Lillehaug, A., A. Ramstad, K. Baekken, L.J. Reitan. 1993. Protective immunity in Atlantic salmon (Salmo salarL.) vaccinated at different water temperatures. Fish Shellfish Immunol. 3: 143-156. Lindsay, G.J.H. 1986. The significance of chitinolytic enzymes and lysozyme in rainbow trout (Salmo gairdneri) defence. Aquaculture. 51: 169-173. Little, M., and S. Keller. 1987. A British Columbia perspective on salmon farming. Alaska Sea Grant Rep. 88: 135-142. McCarthy, D.H. 1983. An experimental model for fish furunculosis caused by Aeromonas salmonicida. J. Fish. Dis. 6: 231-237. McCarthy, D.H., D.F Amend, K.A. Johnson, and J.V. Bloom. 1983. Aeromonas salmonicida : Determination of an antigen associated with protective immunity and evaluation of an experimental bacterin. J. Fish Dis. 6:155-174. McLeay, D.J., and D.A. Brown. 1974. Growth stimulation and biochemical changes in juvenile coho salmon (Oncorhynchus kisutch) exposed to bleach kraft pulpmill effluent for 200 days. J. Fish. Res. Board Can. 31: 1043-1049. McLeay, D.J., and M.R. Gordon. 1977. Leucocrit: a simple hematological technique for measuring acute stress in salmonid fish, including stressful concentrations of pulpmill effluent. J. Fish. Res. Board Can. 34: 2164-2175. Manning, M.J., and Mughal, M.S. 1985. Factors affecting the immune responses of immature fish. p. 27-37 In A.E. Ellis (ed.). Fish and shellfish pathology. Academic Press Inc., London. Matsuyama, H., R.E.P. Mangindaan, and T. Yano. 1992. Positive effects of schizophyllan and scleroglucan against Streptococcus sp. infection in yellowtail (Seriola quinqueradiata). Aquaculture. 101: 197-203. 76  Maule, A.G., and C.B. Schreck. 1990. Changes in number of leukocytes in immune organs of juvenile coho salmon after acute stress or Cortisol treatment. J. Aquat. Anim. Health. 2: 298-304. Maule, A.G., R.A. Tripp, S.L. Kaattari, and C.B. Schreck. 1989. Stress alters immune function and disease resistance in chinook salmon (Oncorhynchus tshawtscha). J. Endocrinol. 120: 135-142. Michel, C. 1982. Progress towards furunculosis vaccination, p. 151-170 In R.J. Roberts (ed.). Microbial diseases offish. Academic Press Inc., London. Michel, C. 1980. A standardized model of experimental furunculosis in rainbow trout (Salmo gairdneri). Can. J. Fish. Aquat. Sci. 37: 746-750. Michel, C , and B. Faivre. 1982. Occurrence and significance of agglutinating antibodies in experimental furunculosis of rainbow trout, Salmo gairdneri Richardson. J. Fish Dis. 5: 429-432. Midtlyng, P.J., M. Carrillo, L. Dahle., J. Morales, P. Sorgeloos, N. Svennevig, J. Wyban. 1993. (eds.) Efficacy and side effects in Atlantic salmon (Salmo salar L.) of injectible furunculosis vaccines. From Discovery to Commercialization. Spec. Publ. Eur. Aquacult. Soc. 19: 414 Summary only. Mitchell, H.M. 1992. Furunculosis treatment, control and chemotherapeutic regulatory approaches to salmon aquaculture in the United States. Bull. Aquacult. Assoc. Can. 92: 22-28. Mock, A., and Peters, G. 1990. Lysozyme activity in rainbow trout, Oncorhynchus mykiss (Walbaum), stressed by handling, transport and water pollution. J. Fish. Biol. 37: 873-885. Moeyner, K., K.H. Reed, S. Sevatdal, and M. Heum. 1993. Changes in non-specific immune parameters in Atlantic salmon Salmo salar L., induced by Aeromonas salmonicida infection. Fish Shellfish Immunol. 3: 253-265. Moldoveanu, Z., M. Novak, Wen-Qiang Huang, R.M. Gilley, J.K. Staas, D. Schafer, R.W. Compans, and J. Mestecky. 1993. Oral immunization with influenza virus in biodegradable microspheres. J. Infect. Dis. 167: 84-90. Moore-Clark Co. Canada Inc. 1993. Royal response. Feeding for health. Sales pamphlet. Vancouver, British Columbia. Morgan, J.D., and G.K. Iwama. 1991. Effects of salinity on growth, metabolism, and ion regulation in juvenile rainbow trout (Salmo gairdneri) and fall chinook salmon (Oncorhynchus tshawytscha). Can. J. Fish. Aquat. Sci. 48: 20832094. Morgan, J.D., and G.K. Iwama. 1993. Stress assessment manual. Simple field methods for the assessment of stress and general condition of salmonid fish. Department of Animal Science, University of British Columbia. 34 p. 77  Morris, W., M.C. Steinhoff, and P.K. Russell. 1994. Potential of polymer microencapsulation technology for vaccine innovation. Vaccine. 12: 5-11. Mulcahy, G. and P.J. Quinn. 1986. A review of immunomodulators and their application in veterinary medicine. J. Vet. Pharmacol. Therap. 9:119-139. Muona, M., and A. Soivio. 1992. Changes in plasma lysozyme and blood leucocyte levels of hatchery-reared Atlantic salmon (Salmo salar L.) and sea trout (Salmo trutta L.) during parr-smolt transformation. Aquaculture. 106: 75-87. Newman, S.G., and J.J. Majnarich. 1985. Immunization of salmonids against furunculosis. Fish Pathol. 20: 403-411. Nikl, L, L.J. Albright, and T.P.T. Evelyn. 1991. Influence of seven immunostimulants on the immune response of coho salmon to Aeromonas salmonicida. Dis. Aquat. Org. 12: 7-12. Nikl, L., Evelyn, T.P.T., and Albright, L.J. 1993. Trials with an orally and immersionadministered beta-1,3 glucan as an immunoprophylactic against Aeromonas salmonicida in juvenile chinook salmon Oncorhynchus tshawytscha. Dis. Aquat. Org. 17: 191-196. Nonaka, M., N. Yamaguchi, S. Natsuume-Sakai, and M. Takahashi. 1981. The complement system of rainbow trout (Salmo gairdneri) I. Identification of the serum lytic system homologous to mammalian complement. J. Immunol. 126: 1495-1498. Olivier, G. 1990. Virulence of Aeromonas salmonicida: Lack of relationship with phenotypic characteristics. J. Aquat. Anim. Health 2:119-127. Olivier, G., T.P.T. Evelyn, and R. Lallier. 1985. Immunogenicity of vaccines from a virulent and an avirulent strain of Aeromonas salmonicida. J. Fish Dis. 8: 43-55. Paterson, W.D., D. Airdrie, and J.L. Zinn. 1988. Field performances of aqua-health furunculosis vaccines. Proc. Aquacult. Int. Congr. p. 59. Summary only. Peters, G., and R. Schwarzer. 1985. Changes in hemopoietic tissue of rainbow trout under influence of stress. Dis. Aquat. Org. 1: 1-10. Pickering, A.D., T.G. Pottinger, R.E. Peter, M.P. Schreibman, P.K.T. Pang, and J.F. Leatherland (eds.). 1988 Stress responses and disease resistance in salmonid fish: Effects of chronic elevation of plasma Cortisol. Fish Physiol. Biochem. 7: 253-258. Plata-Salaman, C.R., and J.P. Borkoski. 1993. Centrally administered bacterial lipopolysaccharide depresses feeding in rats. Pharmacol. Biochem. Behav. 46: 787791. Post, G. 1987. Textbook offish health. T.F.H. Publications, Inc., Neptune City, New Jersey. 288 p.  78  Ray, R., M. Novak, J.D. Duncan, Y. Matsuoka, and R.W. Compans. 1993. Microencapsulated human parainfluenza virus induces a protective immune response. J. Infect. Dis. 167: 752-755. Rice, J.A. 1990. Bioenergetics modeling approaches to evaluation of stress in fish. American Fisheries Society Symposium 8: 80-92. Ricker, W.E. 1975. Computation and interpretation of biological statistics in fish populations. Bull. Fish. Res. Board Can. 191: 382 p. Ricker, W.E. 1979. Growth rates and models, p. 677-743 In W. Hoar and D.J. Randall (eds.). Fish physiology. Vol. VIII Academic Press Inc., London. Roberson, B.S. 1990. Bacterial agglutination, p. 81-86 In J.S. Stolen, T.C. Fletcher, D.P. Anderson, B.S. Roberson, W.B. van Muiswinkel (eds.). Techniques in fish immunology -1. SOS Publications, Fair Haven, New Jersey. Rodgers, C.J. 1990. Immersion vaccination for control offish furunculosis. Dis. Aquat. Org. 8: 69-72. Rodgers, C.J. and B. Austin. 1984. Oral immunisation against furunculosis: An evaluation of two field trials, p. 185-194 \n M.J. Manning and M.F. Tatner (eds.). Fish immunology. Academic Press Inc., London. Rombough, P.J. 1988. Growth, aerobic metabolism, and dissolved oxygen requirements of embryos and alevins of steelhead trout, (Salmo gairdneri). Can. J. Zool. 66: 651660. Sakai, D.K. 1981. Spontaneous and antibody-dependent hemolysis activities offish sera and inapplicability of mammalian complements to the immune hemolysis reaction of fishes. Bull. Jap. Soc. Sci. Fish. 47: 979-991. Sakai, D.K. 1982. Determination of health status and non-specific complement activities in the sera of salmonid fishes. Bull. Eur. Ass. Fish Path. 2: 32-34. Sakai, D.K. 1983. Lytic and bactericidal properties of salmonid sera. J. Fish Biol. 23: 457466. Sakai, D.K. 1984. The non-specific activation of rainbow trout, Salmo gairdneri Richardson, complement by Aeromonas salmonicida extracellular products and the correlation of complement activity with the inactivation of lethal toxicity products. J. Fish Dis. 7: 329-338. Schreck, C.B. 1990. Physiological, behavioral, and performance indicators of stress. American Fisheries Society Symposium 8: 29-37. Schreck, C.B., A.G. Maule, and CH. Slater. 1991. Stress affects the immune response and health of fish in aquacultural systems. Program and Abstracts. Second International Marine Biotechnology Conference (IMBC '91). p. 58. Abstract only. 79  Sheppard, M.E. 1992. Clinical impressions of furunculosis in British Columbian waters. Bull. Aquacult. Assoc. Can. 92: 29-30. Siwicki, A. 1987. Immunomodulating activity of levamisole in carp spawners, Cyprinus carpio L. J. Fish. Biol. 31 (Supplement A): 245-246. Siwicki, A.K., and D.P. Anderson. 1993. Immunostimulation in fish: measuring the effects of stimulants by serological and immunological methods. Abstract and techniques manual presented at The Nordic Symposium on Fish Immunology, Lysekil, Sweden. 19-22 May 1993. 25 pp. Siwicki, A.K., D.P. Anderson, and O.W. Dixon. 1990. In vitro immunostimulation of rainbow trout (Oncorhynchus mykiss) spleen cells with levamisole. Dev . Comp. Immunol. 14: 231-237. Spence, K.D., J.L. Fryer, and K.S. Pilcher. 1965. Active and passive immunization of certain salmonid fishes against Aeromonas salmonicida. Can. J. Micro. 11: 397-405. Spence, J.A., D. Egan, M. Evans, R.H. Cook, and W. Pennell (eds.). 1989. The present status and future prospects of salmon farming in British Columbia. Can. Tech. Rep. Fish. Aquat. Sci. 1831: 37-50. Stoffregen, D.A., A.J. Chako, S. Backman, and J.G. Babish. 1993. Successful therapy of furunculosis in Atlantic salmon, Salmo salar L., using the fluoroquinolone antimicrobial agent enrofloxacin. J. Fish Dis. 16: 219-228. Studnicka, M., A. Siwicki, and B. Ryka. 1986. Lysozyme level in carp (Cyprinus carpio L.). Bamidgeh. 38: 22-25. Sumpter, J.P. 1992. The deleterious effects of stress and their significance to aquaculture. Spec. Publ. Eur. Aquacult. Soc. 18: 157-166 Tatner, M.F., A. Adams, and W. Leschen. 1987. An analysis of the primary and secondary antibody response in intact and thymectomized rainbow trout (Salmo gairdneri) to human gamma globulin and Aeromonas salmonicida. J. Fish. Biol. 31: 177-195. Thornton, J.C, R.A. Garduno, and W.W. Kay. 1992. An attenuated mutant oi Aeromonas salmonicida as a live vaccine for salmonid furunculosis. Bull. Aquacult. Assoc. Can. 92: 53-57. Thornton, J.C, R.A. Garduno, S.G. Newman, and W.W. Kay. 1991. Surface-disorganized, attenuated mutants oi Aeromonas salmonicida as furunculosis live vaccines. Microb. Pathog. 11: 85-99. Thuvander, A., U.P. Wichardt, and L.J. Reitan. 1993. Humoral antibody response of brown trout Salmo trutta vaccinated against furunculosis. Dis. Aquat. Org. 17:17-23. Udey, L.R., and J.L. Fryer. 1978. Immunization of fish with bacterins of Aeromonas salmonicida. Marine Fish. Rev. 40: 12-17. 80  Vander, A.J., J.H. Sherman, and D.S. Luciano (eds.). 1990. Human physiology, the mechanisms of body function, fifth edition. McGraw-Hill Pub. Co. New York, NY. 724 P-  Velji, M.I., L.J. Albright, and T.P.T. Evelyn. 1992. Immunogenicity of various Vibrio ordalii lipopolysaccharide fractions in coho salmon Oncorhychus kisutch. Dis. Aquat. Org. 12: 97-101. Wang, K., L. Waselenchuk, and M.D. Evered. 1993. Stimulation of drinking by bacterial endotoxins in the rat. Physiol. Behav. 54: 1005-1009. Wedemeyer, G.A. 1976. Physiological response of juvenile coho salmon (Oncorhynchus kisutch) and rainbow trout (Salmo gairdneri) to handling and crowding stress in intensive fish culture. J. Fish. Res. Board Can. 33: 2699-2702. Wedemeyer, G.A., B.A. Barton, and D.J. McLeay. 1990. Stress and acclimation, p. 451-489 In C.B. Schreck and P.B. Moyle (eds.). Methods for fish biology. American Fisheries Society, Bethseda, Maryland. Wedemeyer, G.A., R.W. Gould, and W.Y. Yasutake. 1983. Some potentials and limits of the leucocrit test as a fish health assessment method. J. Fish Biol. 23: 711-716. Wedemeyer, G.A., and D.J. McLeay. 1981. Methods for determining the tolerance of fishes to environmental stressors, p. 247-275 In A.D. Pickering (ed). Stress and fish. Academic Press Inc., London. Weir, D.M. (ed.). 1986. Handbook of experimental immunology in four volumes, 4th edition. Blackwell Scientific Publications, Oxford. Volume 1, p. 5A.1 - 5A.23. Yano, T. 1992. Assays of hemolytic complement activity, p. 131-141. In J.S. Stolen, T.C. Fletcher, D.P. Anderson, S.L. Kaattari and A.F. Rowley (eds.). Techniques in fish immunology - 2. SOS Publications, Fair Haven, New Jersey. Yano, T., R.E.P. Mangindaan, and H. Matsuyama. 1989. Enhancement of the resistance of carp Cyprinus carpio to experimental Edwardsiella tarda infection, by some P-1,3glucans. Bull. Jap. Soc. Sci. Fish. 55: 1815-1819.  81  APPENDIX. Data tables.  82  Table 5.  Mean (± 1 SE) condition factors of juvenile rainbow trout at three different times following immunization with seven different A salmonicidavaccines.  Treatment group  Time (weeks post vaccination)  Sample size (n)  Condition factor (mean)  Saline  5  80  1.74 ± 0 . 0 1  12  82  1.71 ± 0 . 0 2  24  37  1.68 ± 0 . 0 9  5  80  1.74 ± 0 . 0 5  12  78  1.66 ± 0 . 0 3  24  35  1.85±0.13  5  80  1.89 ± 0 . 1 5  12  86  1.66 ± 0 . 0 3  24  33  1.73 ± 0 . 0 1  5  80  1.76 ± 0 . 0 3  12  80  1.67 ± 0 . 0 4  24  70  1.63 ± 0 . 0 3  5  80  1.74 ± 0 . 0 1  12  82  1.71 ± 0 . 0 5  24  69  1.65 ± 0 . 0 1  5  80  1.78 ± 0 . 0 5  12  83  1.68 ± 0 . 0 2  24  68  1.68 ± 0 . 0 2  5  80  1.95±0.15  12  82  1.69 ± 0 . 1 1  24  33  1.84 ± 0 . 1 5  5  80  1.80 ± 0 . 0 5  12  83  1.65 ± 0 . 0 5  24  64  1.68 ± 0 . 0 2  Bacterin  Bacterin with levamisole  Oil adjuvanted bacterin  Microencapsulated bacterin  Microencapsulated bacterin with muramyl dipeptide  Microencapsulated bacterin with (3-1,3 glucan  Microencapsulated bacterin with V. anguillarum LPS  83  Table 6.  Mean (+ 1 SE) specific growth rates of juvenile rainbow trout at three different times following immunization with seven different A salmonicida vaccines.  Treatment group Saline  Bacterin  Bacterin with levamisole  Oil adjuvanted bacterin  Microencapsulated bacterin  Microencapsulated bacterin with muramyl dipeptide  Microencapsulated bacterin with p-1,3 glucan  Microencapsulated bacterin with V. anguillarum LPS  Time (weeks post vaccination) 5  Sample size (n) 80  Specific growth rate (% wt per day)  12  82  1.34 + 0.08  24 5  37 80  1.08 + 0.05 1.09 ±0.05  12  78  1.32 + 0.01  24 5  35 80  1.03 ±0.03 1.28 ±0.06  12  86  1.35 ± 0.11  24 5  33 80  1.10±0.04 1.36 ±0.03  12  80  1.38 ±0.16  24 5  70 80  1.06 ±0.02 1.37 ±0.03  12  82  1.38 ±0.10  24 5  69 80  1.02 ±0.00 1.49 ±0.14  12  83  1.37 ±0.02  24 5  68 80  1.05 ±0.02 1.31 ±0.09  12  82  1.36 ±0.20  24 5  33 80  1.08 ±0.02 1.76 ±0.09  12  83  1.42 ±0.12  24  64  1.05 ±0.01  84  1.06 ±0.01  Table 7.  Mean (± 1 SE) oxygen consumption rates of juvenile rainbow trout at three different times following immunization with seven different A salmonicida vaccines.  Treatment group Saline  Bacterin  Bacterin with levamisole  Oil adjuvanted bacterin  Microencapsulated bacterin  Microencapsulated bacterin with muramyl dipeptide Microencapsulated bacterin with p-1,3 glucan  Microencapsulated bacterin with V. anguillarum LPS  Time of sampling (weeks)  Metabolic Rate (mg 0 /kg/h)  3  Sample size (n) 6  6  6  338.6 ±21.7  12 3  6 6  364.9 ± 28.7 311.6 ±24.9  6  6  329.1 ±21.1  12 3  6 6  399.1 ±26.0 401.9 ±24.9  6  6  368.8 ±20.1  12 3  6 6  393.3 ± 33.8 400.1 ±26.6  6  6  354.9 ±41.0  12 3  6 6  327.1 ±31.8 365.1 ±49.7  6  6  342.0 + 13.4  12 3  6 6  416.1 ± 17.1 329.8 ± 39.9  6  6  330.6 ± 24.9  12 3  6 6-  381.2 ±29.8 336.0 ± 35.2  6  6  353.1 ± 19.2  12 3  6 6  407.2 ±22.5 512.5±41.7  6  6  325.3 ±36.0  12  6  341.9 ±30.9  85  2  318.8 ±22.4  Table 8.  Mean (± 1 SE) haematocrit values (% RBC) in juvenile rainbow trout at three different times following immunization with seven different A salmonicida vaccines.  Treatment group Saline  Bacterin  Bacterin with levamisole  Oil adjuvanted bacterin  Microencapsulated bacterin  Microencapsulated bacterin with muramyl dipeptide Microencapsulated bacterin with P-1,3 glucan Microencapsulated bacterin with V. anguillarum LPS  Time (weeks post vaccination) 5  Sample size (n) 20  Haematocrit (packed RBC's as % volume) 39.0 ± 1.11  12  24  42.0 ± 0.79  24 5  10 20  44.2 ± 1.38 37.1 ± 1.20  12  24  40.1 ±0.62  24 5  10 20  45.2 ± 1.55 37.3± 1.19  12  24  40.5 ± 0.79  24 5  10 20  44.5 ± 1.09 36.9 ± 1.41  12  24 20  41.6 ±0.94  24 5  20  45.1 ±0.75 40.1 ±1.33  12  24  40.3 ± 0.76  24 5  20 20  46.5 ± 1.35 40.4 ±1.31  12  24  41.0 ±0.77  24 5  20 20  46.2 ± 0.86 37.5±1.19  12  24  41.5 ±0.94  24 5  20 20  48.6 ± 1.14 45.3 ± 1.42  12  24  42.2 ±1.45  24  10  43.1 ±0.94  86  Table 9.  Mean (± 1 SE) leucocrit values and total leucocytes in juvenile rainbow trout blood at three different times following immunization with seven different A. salmonicida vaccines.  Treatment group Saline  Bacterin  Time Sample (weeks post size vaccination) (n) 20 5  Leucocrit (WBC's as % volume) 1.61 ±0.14  Leucocytes as % of total blood cells  12  24  1.40 ±0.09  2.19±0.15  24 5  10 24 12  1.29 ±0.05 1.30 ±0.17  1.65 ±0.21 3.56 ± 0.35  1.30 ±0.08  2.07 ±0.15  12 10 Bacterin with levamisole  Oil adjuvanted bacterin  Microencapsulated bacterin  Microencapsulated bacterin with muramyl dipeptide Microencapsulated bacterin with p-1,3 glucan Microencapsulated bacterin with V. anguillarum LPS  3.26 ± 0.31  24 5  20  1.49 ±0.06 1.75 ±0.11  2.38 ± 0.29 2.67 ± 0.23  12  24  1.46 ±0.04  1.90±0.13  24 5  10 20  1.34 ±0.08 1.60 ±0.10  2.05 ± 0.20 3.49 ± 0.23  12  24 20  1.54 ±0.09  2.18±0.14  24 5  20  1.72 ±0.06 1.54 ±0.14  2.53 ± 0.20 3.22 ± 0.36  12  24  1.24 ±0.07  2.01 ±0.14  24 5  20 20  1.59±0.10 1.54 ±0.13  2.16 ±0.26 4.24 ± 0.40  12  24  1.29 ±0.09  2.21 ±0.14  24 5  20 20  1.64 ±0.09 1.73 ±0.16  2.34 ±0.16 5.76 ± 0.99  12  24 .  1.16 ±0.05  1.81 ±0.18  24 5  10 20  1.76 ±0.08 1.59 ±0.11  2.22 ±0.16 2.80 ± 0.23  12  24  0.77 ±0.07  2.31 ±0.18  24  20  1.54 ±0.11  2.70 ±0.15  87  Table 10. Mean (± 1 SE) percentage of lymphocytes and neutrophils in juvenile rainbow trout blood at three different times following immunization with seven different A salmonicida vaccines. Treatment group Saline  Bacterin  Bacterin with levamisole  Oil adjuvanted bacterin  Microencapsulated bacterin  Microencapsulated bacterin with muramyl dipeptide  Microencapsulated bacterin with p-1,3 glucan  Microencapsulated bacterin with V. anguiliarum LPS  Time Sample (weeks post size vaccination) (n) 5 20  Lymphocytes as % WBC's  Neutrophils as % WBC's  94.1 ± 1.65  5.09 ±1.14  12  24  90.2 ± 1.89  4.19 ±0.86  24 5  20 24  93.7 ±2.20 94.2 ±1 .39.  3.41 ±1.08 5.87 ±1.31  12  12  82.9 ± 2.74  4.32 ± 0.88  24 5  20 20  93.2 ± 2.06 93.1 ±1.80  2.90 ± 0.92 5.15 + 1.15  12  24  85.5 ±2.70  4.84 ± 0.99  24 5  20 20  95.0 ±1.90 92.3 ± 1.41  4.55 ± 1.44 3.87 ± 0.86  12  24  89.3 ±2.47  4.24 ± 0.87  24 5  20 20  90.6 ± 2.45 90.4 ±1.63  5.48 ±1.23 5.90 ±1.32  12  24  86.2 ± 3.02  3.49 ± 0.71  24 5  20 20  93.6 ± 1.32 90.4 ± 1.93  3.20 ± 0.72 6.11 ±1.37  12  24  91.3 ±1.37  4.79 ± 0.98  24 5  20 20  85.5 ± 2.48 88.2 ± 2.27  4.17 ±0.93 6.24 ±1.40  12  24  89.3 ± 1.72  3.55 ± 0.72  24 5  20 20  91.8 ±2.49 94.2 ±1.52  3.46 + 1.09 3.76 ± 0.84  12  24  93.2 ±1.27  3.40 ± 0.69  24  20  93.7 ± 1.01  1.90 + 0.43  88  Table 11. Mean (± 1 SE) percentage of thrombocytes and monocytes in juvenile rainbow trout blood at three different times following immunization with seven different A salmonicida vaccines. Treatment group Saline  Bacterin  Bacterin with levamisole  Oil adjuvanted bacterin  Microencapsulated bacterin  Microencapsulated bacterin with muramyl dipeptide  Microencapsulated bacterin with P-1,3 glucan Microencapsulated bacterin with V. anguillarum LPS  Time Sample (weeks post size vaccination) (n) 5 20  Thrombocytes as % WBC's  Monocytes as % WBC's  2.19 + 0.79  0.60 ± 0.45  12  24  4.98 ±1.30  4.46 ± 1.49  24 5  10 24  4.74 ±2.01 1.28 ±0.52  0.00 ±0.00 0.64 ± 0.46  12  12  8.75 ±1.23  1.44 ±0.61  24 5  10 20  3.72 ±1.73 3.77 ± 1.34  0.67 ± 0.67 0.66 ± 0.46  12  24  9.15 ± 1.99  2.18 ±0.83  24 5  10 20  3.19 ±1.57 3.66 ±1.08  0.00 ±0.00 2.29 ± 0.65  12  24  7.89 ±2.18  0.90 ± 0.45  24 5  20 20  6.39 ± 1.51 3.66 + 1.56  0.00 ± 0.00 0.63 + 0.63  12  24  9.80 ±2.05  0.73 ±0.41  24 5  20 20  4.38 ± 1.27 4.43 ±1.46  0.55 ±0.43 0.88 ± 0.39  12  24  4.66 ±1.19  1.17 ±0.70  24 5  20 20  9.75 ±2.11 5.36 ±1.61  1.75 ±0.72 2.29 ±1.02  12  24  7.88 ±1.52  1.41 ±0.61  24 5  10 20  2.89 ±1.99 2.10 ±0.94  2.01 ±0.83 0.82 ± 0.64  12  24  3.62 ±1.05  1.56 ±0.60  24  20  4.04 ± 0.94  1.25 ±0.45  89  Table 12. Mean (± 1 SE) somatic index of the head kidney and total kidney in juvenile rainbow trout at three different times following immunization with seven different A. salmonicida vaccines. Treatment group Saline  Bacterin  Bacterin with levamisole  Oil adjuvanted bacterin  Microencapsulated bacterin  Microencapsulated bacterin with muramyl dipeptide  Microencapsulated bacterin with P-1,3 glucan Microencapsulated bacterin with V. anguillarum LPS  Time (weeks post vaccination) 5  Sample size (n) 20  Head Kidney (% body weight) 0.250 ±0.015  Total Kidney (% body weight) 0.896 ± 0.028  12  24  0.281 ±0.011  0.934 ± 0.022  24 5  10 24  0.207 ±0.012 0.236 ±0.015  0.787 ± 0.026 0.926 ± 0.028  12  12  0.279 ± 0.009  0.935 ±0.013  24 5  10 20  0.235 + 0.012 0.264± 0.012  0.884 ± 0.031 0.958 ± 0.024  12  24  0.290 ± 0.009  0.972 ± 0.021  24 5  10 20  0.226 + 0.013 0.249 ±0.012  0.883 ± 0.044 0.969 ± 0.026  12  24 20  0.281 ±0.013  0.926 ± 0.021 0.870 ± 0.024 0.927± 0.023  24 5  20  0.261 ±0.013 0.223 ±0.010  12  24  0.261 ±0.010  0.898 ± 0.019  24 5  20 20  0.243 ±0.140 0.210 ±0.012  0.863 ± 0.026 0.964 ± 0.026  12  24  0.306 ±0.013  0.905 ± 0.024  24 5  20 20  0.302 ± 0.022 0.213 ±0.011  1.016 ±0.034 0.916 ±0.029  12  24  0.237 ±0.011  0.852 ± 0.021  24 5  10 20  0.240 ±0.015 0.210 ±0.009  0.859 ± 0.022 0.932 ± 0.022  12  24  0.244 ±0.014  0.843 ± 0.021  24  20  0.439 ±0.125  1.051 ±0.050  90  Table 13. Mean (± 1 SE) somatic index of spleen tissue in juvenile rainbow trout, immunized with seven different A salmonicida vaccines, at three different times. Treatment group Saline  Bacterin  Bacterin with levamisole  Oil adjuvanted bacterin  Microencapsulated bacterin  Microencapsulated bacterin with muramyl dipeptide  Microencapsulated bacterin with P-1,3 glucan Microencapsulated bacterin with V. anguillarum LPS  Time (weeks post vaccination) 5  Sample size (n) 20  Spleen (% body weight)  12  24  0.144 + 0.007  24 5  10 24  0.122 ±0.015 0.112 ±0.007  12  12  0.174 ±0.011  24 5  10 20  0.113±0.010 0.119 ±0.007  12  24  0.180 ±0.010  24 5  10 20  0.121 ±0.016 0.096 ± 0.006  12  24 20  0.172 ±0.014  0.108 ±0.006  24 5  20  0.106 ±0.007 0.110 ±0.008  12  24  0.190 ±0.012  24 5  20 20  0.120 ±0.007 0.111 ±0.009  12  24  0.171 ±0.010  24 5  20 20  0.121 ±0.011 0.114 ±0.008  12  24  0.159 ±0.008  24 5  10 20  0.112 ±0.013 0.111 ±0.009  12  24  0.168 ±0.009  24  20  0.128 ±0.010  91  Table 14. Mean (± 1 SE) kidney lysozyme activity in juvenile rainbow trout at three different times following immunization with seven different A salmonicida vaccines. Values are expressed as units of activity per gram kidney tissue Treatment group Saline  Bacterin  Bacterin with levamisole  Oil adjuvanted bacterin  Microencapsulated bacterin  Microencapsulated bacterin with muramyl dipeptide  Microencapsulated bacterin with P-1,3 glucan Microencapsulated bacterin with V. anguillarum LPS  Time (weeks post vaccination) 5  Sample size (n) 20  Lysozyme activity  12  24  644.4 ± 35.7  24 5  10 24  1344.4 + 102.6 2381 ±199.0  12  12  685.4 ± 46.3  24 5  10 20  902.5 ± 93.7 2743.4 ± 285.3  12  24  720.5 ±59.1  24 5  10 20  1309.8 ± 180.1 1740.4 ± 129.6  12  24  988.5 ± 105.9  24 5  20 20  847.6 ± 67.8 4389.1 ±1205.0  12  24  768.4 ± 46.9  24 5  20 20  1263.8 ± 128.0 3119.1 ±509.4  12  24  965.0 ±101.8  24 5  20 20  1061.4 ±65.4 2406.9 ±218.5  12  24  724.9 ± 55.2  24 5  10 20  1441.3 ±292.0 1883.5 ± 168.3  12  24  897.3 ±88.3  24  20  2154.0 ±532.6  92  (U/g)  1792.7 ±155.5  Table 15. Mean (± 1 SE) agglutinating antibody titres in juvenile rainbow trout at three different times following immunization with seven different A salmonicida vaccines. Values are expressed as the reciprocal of the highest agglutinating dilution. Treatment group Saline  Bacterin  Bacterin with levamisole  Oil adjuvanted bacterin  Microencapsulated bacterin  Microencapsulated bacterin with muramyl dipeptide Microencapsulated bacterin with P-1,3 glucan Microencapsulated bacterin with V. anguillarum LPS  Time (weeks post vaccination) 5  Sample size "(n) 20  Agglutinating antibody titer  12  24  8.00 ± 1.38  24 5  10 24  8.43 ± 2.67 17.90 ±4.29  12  12  122.00 ±33.78  24 5  10 20  211.20 + 40.53 22.21 ± 5.03  12  24  18.33 ±2.64  24 5  10 20  139.20 ±46.74 21.40 ±4.73  12  24  1225.33 ± 170.33  24 5  20 20  267.2 ±69.11 13.70 ±3.95  12  24  95.00 ± 45.73  24 5  20 20  46.80 ± 7.30 22.30 ±5.18  12  24  152.67 ±84.50  24 5  20 20  68.00 ± 12.66 22.44 ± 5.04  12  24  47.33 ± 23.09  24 5  10 20  128.00 ±51.60 41.33 ± 10.63  12  24  146.09 ±47.50  24  20  995.20 ±167.36  93  0.22 ±0.15  Table 16. Mean (± 1 SE) plasma haemolytic activity in immunized juvenile rainbow trout at three different times following immunization with seven different Aeromonas salmonicida vaccines. Treatment group Saline  Bacterin  Bacterin with levamisole  Oil adjuvanted bacterin  Microencapsulated bacterin  Microencapsulated bacterin with muramyl dipeptide  Microencapsulated bacterin with P-1,3 glucan  Microencapsulated bacterin with V. anguillarum LPS  Time (weeks post vaccination) 5  Sample size (n) 20  Haemolytic activity (units)  12  24  6.21 ± 0.744  24 5  10 20  15.0 ±2.769 4.08 ± 0.300  12  24  8.60 ± 0.728  24 5  10 20  11.1 ±1.854 5.88 ± 0.539  12  24  7.71 ±0.651  24 5  10 20  12.2 ± 1.377 8.94 ± 0.486  12  24  6.84 ± 0.685  24 5  20 20  10.9 ± 1.010 8.63 ± 0.390  12  24  6.96 ± 0.828  24 5  20 20  14.4± 1.163 7.66 ± 0.335  12  24  8.36 ± 0.724  24 5  20 20  11..2 + 2.459 7.69 ± 0.370  12  24  9.43 ± 1.012  24 5  10 20  14.5 ±2.459 7.23 ± 0.665  12  24  8.84 ±1.135  24  20  10.2 ± 1.086  94  4.28 ± 0.486  

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
http://iiif.library.ubc.ca/presentation/dsp.831.1-0086740/manifest

Comment

Related Items