COMPARISON OF NATURALLY-ACQUIRED AND VACCINE-ACQUIRED IMMUNITY TO LOMA SALMONAE IN RAINBOW TROUT (ONCORHYNCHUS MYKISS)

BY

JENNIFER E. HARKNESS

A Thesis

Submitted to the Graduate Faculty

in Partial Fulfillment of the Requirements

for the Degree of

MASTER OF SCIENCE

Department of Pathology and Microbiology

Faculty of Veterinary Medicine

University of Prince Edward Island

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REMOVED ABSTRACT

Microsporidial Gill Disease of Salmon (MGDS) is a result of infection withLoma salmonae and has been described in fanned and wild Pacific salmon as causing severe branchitis, resulting in significant economic losses in aquaculture, as fish succumbing to the disease are those nearing market size. No treatment currently exists to controlL. salmonae, although previous studies have documented disease resistance upon initial recovery and secondary exposure to the parasite. The objectives of this study were to further examine naturally acquired as well as vaccine-acquired immunity toL. salmonae in rainbow trout (RBT). It is believed that farm reared salmon are exposed to continual low doses of the parasite over time, and the naturally acquired immunity studies focused on the protective response generated by recovery of a low dose cohabitation exposure and subsequent high dose oral re-challenge. Having the ability to partially protect RBT upon re-exposure toL. salmonae contradicted what is being seen in the netpen scenario, as Chinook and Coho salmon are assumed to have no immunity to re-infection. The investigation into vaccine-acquired immunity focused on the use of heterologous live vaccines, containing Glugea anomala and Glugea hertwigi, respectively. Having common characteristics and being phylogenetically related L.to salmonae, the intraperitoneal (IP) injection into RBT 6 weeks pre­ exposure toL. salmonae was able to reduce xenoma burden significantly as compared to naive controls. Since live vaccines are not marketable, further studies investigated the use of killed heterologous (G. anomala and G. hertwigi) vaccines in comparison with the widely published killed homologous vaccine containing Loma sp. The superiority of both the killed heterologous vaccines were noted, as they were able to reduce xenoma burden upon exposure to the parasite by upwards of 15% compared to the killed homologous vaccine. As a final approach, naturally- acquired immunity was revisited to determine if prophylactic oral treatment of immunostimulant, Provale™, a 0-1,3/1,6 glucan in synergy with low dose recovery could significantly boost the immune response of RBT toL. salmonae. Little evidence was found to support this hypothesis. The immune responses and consequently protection to MGDS in RBT depends greatly upon the intensity of the primary infection and subsequent recovery. Timing of vaccination is essential to the protection of RBT to L. salmonae. Although RBT is a useful surrogate model for infection with MGDS, it does not mimic the exact severity of infection seen in Pacific salmon, particularly Chinook and Coho salmon. With this in mind, experimental studies mimicking those carried out in the thesis in Chinook salmon would be beneficial to the control and treatment of L. salmonae.

v ACKNOWLEDGEMENTS

I wish to thank my supervisor Dr. David Speare for his constant support, creativity, sincerity and encouragement throughout my program. I greatly respect your enthusiasm as a research scientist and supervisor and appreciate your honesty and personal integrity. I would also like to thank the other members of my Supervisory Committee, Drs. Fred Markham, Spencer Greenwood, Collins Kamunde, Fred Kibenge and the members of my Examination Committee, Drs. David Speare, Gary Conboy, Collins Kamunde, Mark Fast and Spencer Greenwood for reviewing and correcting my thesis.

I would still be wandering around the Aquatic Facility staring at empty tanks without the support and encouragement of Dr. Nicole Guselle. As you can imagine, she has had a significant hand in the initialization and continuation of all the experimental aspects of the thesis. In addition, many thanks and gratitude to the AVC Aquatic Facility staff including Wayne Petley, Angela Hamish- Driscoll and Lee Dawson, I couldn’t have achieved this without your tireless work ethics to maintain an amazing facility.

Thanks to Diane MacLean and Rita Saunders for their administrative assistance and Kathy Jones for her photography assistance.

I am extremely grateful to have met so many interesting people over the past few years and wish to thank everyone who has had a hand in the completion of this thesis, whether it was through guidance, having a shoulder to cry on, listening to weekly, if not daily rants of a graduate student, or simply being there in a time of need. To each and every one of you, I thank you for your kindness, support, friendship and love.

Thanks to my parents Donna McKinnon and Michael Poczynek, to my sister Sarah and to the rest of my family for their continued support, encouragement, constructive criticism, and ultimately love. You all hold a special place in my heart.

Special thanks goes to my dearest of friends, Jessica Fry and Angela Hamish-Driscoll. You have both made me a better person and I love you both dearly.

A very large, and very deserving thanks goes to the hundreds of fish that were used in my research, because without them, this thesis would have never been possible. Their sacrifice is truly appreciated and will serve the greater good of the future of aquaculture.

I acknowledge the financial support I received through a Natural Sciences and Engineering Research Council Strategic Grant, the Springboard Atlantic Fund and the Department of Pathology and Microbiology at the Atlantic Veterinary College (UPEI). DEDICATION

To my grandparents,

Della and Burleigh MacKinnon.

Grammy, I hope I’ve made you proud!

Grampy, your love of fish and wildlife resonates in me!

Thank you.

Love Always,

Jenny Penny

vii TABLE OF CONTENTS

Title of Thesis i

Conditions of Use ii

Permission to Use Postgraduate Thesis iii

Certification of Thesis Work iv

Abstract v

Acknowledgements vi

Dedication vii

Table of Contents viii-x

List of Figures xi

List of Tables xii

List of Abbreviations xiii

1. GENERAL INTRODUCTION 1.1 Aquaculture 1 1.1.1 Global Production 1 1.1.2 Canadian Production 2 1.1.3 Salmonid Production 4 1.1.4 Chinook Salmon 5 1.1.5 Netpens 7 1.2 Disease in Netpens 9 1.2.1 Diseases of Chinook Salmon 10 1.2.2 Protistan Diseases of Salmon in Seawater 11 1.3 Microsporidian Parasites 13 1.3.1 Microsporidian Life Cycle 15 1.3.2 Microsporidians in Human and Animal Health 18 1.4 Loma salmonae 20 1.4.1 Transmission 21 1.4.2 Diagnosis and Clinical Signs 22 1.4.3 Immunological Resistance 26 1.4.4 Drug Treatment 29 1.4.5 Vaccines Against Loma salmonae 32 1.5 Research Objectives 33 1.6 REFERENCES 35

2. COMPARATIVE RESISTANCE TO LOMA SALMONAE IN RAINBOW TROUT (ONCORHYNCHUS MYKISS) SUBSEQUENT TO RECOVERY FROM AN INITIAL LOW DOSE COHABITATION EXPOSURE 2.1 ABSTRACT 48 2.2 INTRODUCTION 49 2.3 MATERIALS AND METHODS 52 2.3.1 Sample Population 52 2.3.2 Experimental Design 52 2.3.3 Methods of Infection 53 2.3.4 Challenge Dose Quantification 53 2.3.5 Sampling and Infection Assessment 53 2.3.6 Data Analysis 54 2.4 RESULTS 55 2.5 DISCUSSION 58 2.6 REFERENCES 64

FIRST DEMONSTRATION OF THE PARTIAL EFFICACY OF A HETEROLOGOUS VACCINE AGAINST LOMA SALMONAE , USING SPORES FROM TWO SPECIES OF GLUGEA 3.1 ABSTRACT 68 3.2 INTRODUCTION 69 3.3 MATERIALS AND METHODS 76 3.3.1 Sample Population 76 3.3.2 Experimental Design 77 3.3.3 Methods of Infection 79 3.3.4 Challenge Dose Quantification 79 3.3.5 Sampling and Infection Assessment 80 3.3.6 Data Analysis 80 3.4 RESULTS 80 3.5 DISCUSSION 83 3.6 REFERENCES 86

RESISTANCE TO AN INFECTION WITH LOMA SALMONAE IN RAINBOW TROUT (ONCORHYNCHUS MYKISS) VACCINATED WITH HETEROLOGOUS AND HOMOLOGOUS KILLED SPORES AND WITH OR WITHOUT SUBSEQUENT ORAL ADMINISTRATION OF PROVALE™, A p-1,3/1,6 GLUCAN

4.1 ABSTRACT 91 4.2 INTRODUCTION 92 4.3 MATERIALS AND METHODS 96 4.3.1 Sample Population 96 4.3.2 Experimental Design 97 4.3.3 Methods of Infection 100 4.3.4 Challenge Dose Quantification 101 4.3.5 Sampling and Infection Assessment 101 4.3.6 Data Analysis 102 4.4 RESULTS 102 4.5 DISCUSSION 106 4.6 REFERENCES 110

GENERAL DISCUSSION 114 5.1 REFERENCES 119

APPENDIX A 122 APPENDIX B 123 APPENDIX C APPENDIX D APPENDIX E LIST OF FIGURES

Figure 1. Generalized diagrammatic representation of a microsporidian spore with contents: anchoring disc, polar tube, nucleus, sporoplasm, cell membrane, endospore, exospore and posterior vacuole.

Figure 2. Histological section of rainbow trout gill infected with Loma salmonae. Shown are four large parasitic xenomas containing many spores. H&E. Scale bar = 50 pm

Figure 3. Histological section showing a rupturedLoma salmonae xenoma in the gills of an infected rainbow trout. H&E. Scale bar = 50 pm

Figure 4. Histological section of a Loma salmonae-infected rainbow trout gill showing epithelial hyperplasia. Lamellae show several thickened areas of epithelial tissue. H&E. Scale bar = 50 pm

xi LIST OF TABLES

Table 2.1. The immune response to a high dose oral challenge in rainbow trout that have previously recovered from a low dose cohabitation challenge

Table 3.1. Xenoma outcome at week 5, following experimental challenge with Loma salmonae, in naive vs. vaccinated fish or recovered fish.

Table 4.1. Xenoma outcome at week 5, following an experimental challenge with virulent Loma salmonae, in naive vs. vaccinated Rainbow trout.

Table 4.2. Xenoma outcome at week 5, following an experimental challenge with virulent Loma salmonae, in naive vs. low dose recovered, high dose recovered, P* 1,3/1,6 glucan fed and P-1,3/1,6 glucan fed in synergy with low dose recovery Rainbow trout.

xii LIST OF ABBREVIATIONS

°c -Degrees Celsius fil -Microlitre pm -Micrometre % -Percent AGD -Amoebic Gill Disease DNA -Deoxyribonucleic acid DCH -Dicyclohexamine FIA -Freund's incomplete adjuvant Fig. -Figure ERM -Enteric Red Mouth g -Grams HPAI -Highly Pathogenic Avian Influenza Virus Hsp70 -Heat shock protein 70 i.e. -Id est; that is IHNV -Infectious Hematopoeitic Necrosis Virus IL-12 -Interleukin 12 IP -Intraperitoneal KBMA -Killed but metabolically active kg -Kilograms L -Litre lbs -Pounds m -Metre Mb -Megabase mg -Milligrams MGDS -Microsporidial Gill Disease of Salmon min -Minutes ml -Millilitres MNC -Mononuclear cells OA -Oncorhynchus associated P -P-value PA -Protective antigen PE -Post exposure PCR -Polymerase chain reaction rDNA -Ribosomal deoxyribonucleic acid RNA -Ribonucleic Acid RPS -Relative percentage survival SGR -Specific growth rate SV -Salvelinus variant TLRs -Toll-like receptors TNF-a -Tumor necrosis factor a v/v -Volume for volume XCPGA -Xenoma count per gill arch

xiii 1. GENERAL INTRODUCTION

1.1 Aquaculture

Aquaculture is the farming of aquatic organisms, including finfish and shellfish, by

individuals, groups or corporations using interventions (i.e., feed, medication, controlled breeding, and containment) that enhance production. It is often referred to as “fish farming” and typically involves algae, molluscs, crustaceans and fish (Allain 2005; Bamabe 1990; Sapkota

2008). The demand for global food production is one of the main reasons for large scale culturing of aquatic organisms, but farms also provide fish for re-stocking purposes, hobby farms,

fee-for-fishing ponds, laboratory studies, and ornamental fish (Broussard 1991).

Fish farms are designed not only to meet the demands of the aquatic organism being reared but they also need to be easily accessible by workers as well as efficient at growing out

large, healthy fish that the market demands. Depending upon the species, rearing facilities may be pump-a-shore, ponds, raceways and floating, suspended, or submerged cages (Chua& Tech

2002).

1.1.1 Global Production

Aquaculture from the global perspective began over three thousand years ago when farmers in China began growing fish, particularly Chinese Carp(Mylopharyngodon piceus) in

freshwater ponds. For centuries, aquaculture was a small-scale undertaking that provided relatively low yields (Tibbetts 2001). It was not until the late 1970’s to early 1980’s that aquaculture began to expand at a startling rate. In the past 30 years, global fish consumption rose from 45 million metric tonnes in 1973 to 91 million tons in 1997 (Delgado, Wada, Rosegrant,

Meijer & Ahmed 2003). A portion of this demand is due to increases in global population, particularly in developing countries which rely heavily on fish as a source of protein in their diets.

In response to food shortages and economic issues, the World Bank began encouraging aquaculture in developing countries in order to provide an improved protein supply for consumption and to help generate income (Muir 2005; Tibbetts 2001).

Globally, the major producing region is Asia (84% of global production), while other regions such as Europe (9%) and North America (3%) are making impressive strides (Boghen

1995b). Additionally, aquaculture has grown significantly over the past few decades as 70% of wild fish stocks are estimated to be fully exploited, overexploited, or recovering from a period of depletion (FAO 2004, as cited in Brander 2007). Further demand for global aquaculture production is caused by shifts in consumer dietary habits (Broussard 1991).

In 2004, the combined output of capture fisheries and aquaculture provided the world with 106 million tonnes of food fish, with aquaculture accounting for 43% or approximately 45.6 million tonnes of this total (FAO 2006). The controlled rearing of aquatic organisms continues to grow and is considered the fastest growing animal food-producing sector with a worldwide growth at an average rate of 8.9% per year since 1970, compared with only 1.2% and 2.8% over the same period for capture fisheries and terrestrial farmed meat production systems, respectively

(Subasinghe 2005). It is believed that demands will continue to increase and is estimated that by

2030, more than half of the biomass of fish consumed will come from aquaculture (Tibbetts

2001).

1.1.2 Canadian Production

Compared to the history of global aquaculture production, aquaculture in Canada is relatively new, but nonetheless lucrative. The first documented account of aquaculture in Canada can be traced back to 1857, when the superintendant of fisheries for Lower Canada, Richard

Nettle, studied the incubation and hatching performances of Atlantic salmon (Salmo salar ) and brook trout (Salvelinus fontinalis) eggs. Even before Nettle’s time it is believed that the aboriginal peoples of Canada made early attempts at manipulating salmonid stocks, although

2 supportive documents are relatively scarce (Boghen 1995a). Canada, which benefits from diverse coastlines, paired with a vast array of inland waters and a variety of aquatic resources, has characteristics likely to support considerable growth in aquaculture production (Boghen 1995b).

In 1992, Canada’s contribution to total global aquaculture production in tonnage

(including aquatic plants) was less than 0.2%. Its overall production places Canada twenty-ninth in the world at 30,852 tonnes, in contrast, the total value of its production ranked it twenty-fourth

(ca. $146 million [U.S.]). The ranks differ for various factors including value-added features, quality of the product, and geographic region from where the species originates (Boghen 1995a).

Upon comparison of production values, Canada produced 30,852 tonnes while Chile produced

116,269 tonnes, or about 3.7 times as much as Canada. In terms of value, Chile was worth less than twice as much ($146 million [U.S.] for Canada versus $287 million [U.S.] for Chile)

(Boghen 1995a).

Canada’s limited early involvement in aquaculture could be explained by the minor role of fish and marine plants in the nation’s diet. In the 1980’s, its annual per capita consumption was about 7 kilograms, while in Japan, where fish have an important place in daily cuisine, the consumption rate was five times as much with an annual weight of 35 kilograms (FAO 1984, as cited in Boghen 1995a; Tibbetts 2001).

In 1987, Canadian aquaculture produced 11.5 tonnes of fish valued at $50 million and by

1994 that statistic had risen to an annual amount of46,000 tonnes valued at $300 million (Boghen

1995b). More recent counts of national production levels have shown Canadian finfish and seafood catches weighing in at 160,924 tonnes valued at $919 million with finfish accounting for

122,160 tonnes of this total with a value of $846 million (DFO 2010) (See Appendix A).

Recently, the 2010 Canadian Aquaculture Production Statistics were released showing salmon as the leading species in Canadian aquaculture with 101,385 tonnes of fish produced, accounting for 83% of the total tonnage of finfish production in Canada and 63% of total

Canadian aquaculture (DFO 2010) (See Appendix A).

1.1.3 Salmonid Production

The salmonids of interest in aquaculture are those of the subfamily Salmonidae, which include trout and salmon belonging to the genera Oncorhynchus, Salvelinus and Salmo (Nelson

1976; Laird 1996). These fish are native to the cool water of the northern hemisphere and are anadromous, migrating from the sea in order to spawn in fresh water (Laird 1996).

Sea cage production of large salmonids is relatively new, but is now the dominant technology used in production. The most significant amount of salmonid production takes place in Norway, Chile, United Kingdom, Canada and Japan, with a smaller percentage occurring in

New Zealand and Australia. The dominant species grown is Atlantic salmon {Salmo salar) along with ample amounts of Chinook {Oncorhynchus tshawytscha) and Coho {Oncorhynchus kisutch) salmon. “Steelhead” rainbow trout {Oncorhynchus mykiss), tolerant of marine salinity, are also reared for various local and niche market demands in Japan (Pennell & Barton 1996a).

In the earlier days of the salmonid industry, particularly in Canada, Chinook salmon were the dominant species farmed, although recently, the production of Atlantic salmon has become more extensive due their ability to be stocked at high densities and their flesh marketability. By

1994, 70% of salmon farmed in British Columbia, Canada were Atlantic salmon (Davidson

1999).

Piscivorous species such as salmon are considered one of the most valuable fish products on the market, making international trade a large part of the fanned salmon business (Tibbetts

2001). Norway, Chile, and the United Kingdom are major exporters, while the rest of Europe, the

United States, and Japan are major consumers (Tibbetts 2001).

4 Since the late 1970’s, salmon aquaculture has grown into a tremendous global industiy, producing over one million tonnes of salmon per year (ICES 2006, as cited in Ford & Myers

2008). Additionally, commercial fisheries are becoming increasingly reliant on salmonid hatchery and production programs to supplement depleting stocks. Demands on salmonid production have lead to farmed salmon and trout currently accounting for almost one-half of the total salmonid aquaculture production in the entire world (Pennell et al. 1996a).

1.1.4 Chinook Salmon

Chinook salmon (O. tshawystcha) is one of the seven species of the genus Oncorhynchus native to North America (COSEWIC 2006). Brood fish are of large size and handling these fish during stripping of gametes is typically facilitated by anaesthesia. Because Chinook salmon is a semelparous species they are usually euthanized before stripping (Billard & Jensen 1996).

The most common method of artificial fertilization in Chinook salmon involves the ova being extruded by means of an incision. The abdomen wall of a female fish is cut and ova collected while discarding any coelomic fluid (Billard et al. 1996). Milt is removed from one or more males by applying pressure to the wall of the abdomen and releasing sperm through the genital pore (Billard 1990; Billard et al. 1996). The milt is spread over the ova and a feather is used to mix the gametes, followed by the addition of fresh water (Billard 1990).

Recent technical advances allow for the reduction of the amount of milt used in the artificial fertilization process. Traditional methods employed to examine sperm quality have been based upon sperm viability and membrane integrity, as well as motility, morphology and ultrastructure but have been at best semi-quantitave. Novel quantitative assessments employ the use of computer models to assess sperm characteristics by means of photomicrography and video- micrography techniques (Rurangwa, Kime, Ollevier & Nash 2004). These advanced techniques help to reduce any waste of resources during fertilization. Typically, gametes are stripped from

5 both fish in the traditional manner, but the sperm is stored at 4°C. A diluent, buffered saline solution consisting of 5.54 g/L NaCl, 2.42 g/L tris and 3.75 g/L glycol is spread over the ova, with one litre of diluting solution being enough to cover three litres of ova. Chilled sperm is added to the ova and diluent solution using a syringe at a rate of 3 ml/L. Two decantations are required, followed by the solution sitting for fifteen minutes (Billard 1990).

Eggs are incubated in trays, troughs or boxes with water constantly flowing at an optimum temperature of 12-14°C (Billard 1990). The rate of development during incubation is directly related to water temperature and environmental conditions. Increased water temperature, with consideration of upper lethal levels and abundant dissolved oxygen leads to faster development (Pennell et al. 1996b). It is absolutely necessary to ensure water being delivered to incubators is saturated with oxygen and the flow rate should be about 5 m3 per day for 10,000 eggs on a rack. Oxygen saturation and flow rates need to increase as development progresses in order to meet the embryonic demands (Billard 1990). Additional parameters that require monitoring during embryonic development are total gas pressure, ammonia, pH, carbon dioxide, suspended solids, minerals, and water velocity (Billard et al. 1996).

After 30-35 days of incubation, the eyes are clearly visible through the egg capsule and the embryos are considered to be at the eyed stage and handling at this time is less likely to cause damage (Peterson, Spinney & Sreedharan 1977). Deliberate shocking is used to separate viable and non-viable eggs (Saunders 1995).

Once hatched, the young Chinook salmon are kept in 14-15°C water, at which time they are considered alevins, sustaining themselves on their attached yolk sac. (Saunders 1995; M.

Tchipeff, Creative Salmon Inc. 2008, personal communication). Once the yolk sac is partially absorbed, the fish gulp the surface of the water, filling their swimbladders with oxygen, at which point the fish are considered swim-up or first feeding firy (Saunders 1995). Prepared feed is

6 delivered to these fish at the rearing stations and after a 2-month period the fish are placed into tanks held at 10°C (M. Tchipeff, Creative Salmon Inc. 2008, personal communication). By May of the hatching year the young salmon are considered smoltified, weighing about 25 grams and are transferred to small seawater netpens. By mid-September the fish weigh about 300-400 grams and are transferred to larger netpens where they will continue to grow until first harvest in mid-April, approximately 10-11 months after their initial saltwater entry where they will have reached a dressed weight of up to 2.9 kilograms. In order to satisfy further market demands, a second harvest is typically performed mid-August, approximately 14-15 months after their introduction to saltwater, with a larger dressed weight of 3.0-4.0 kilograms (M. Tchipeff, Creative

Salmon Inc. 2008, personal communication).

1.1.5 Netpens

Chinook salmon production systems in seawater consist of netpens that range from 750 to

2000 m3 and typically are in groups of six or more called a “flotilla” (Munro 1990). The basic marine cage consists of a perimeter structure from which an enclosed cage is suspended in order to hold fish. The two basic types of cage systems are either, floating (the most common), or submerged (Novotny & Pennell 1996). The suspended enclosure can be cuboidal, hemispherical, cylindrical, or conical in shape and is constructed of mesh material, typically soft, knotless nylon webbing, large enough to allow passage of water, but small enough to prevent the escape of the smallest fish within the pen (Novotny et al. 1996). Marine cages are normally accessible by watercraft and are in reasonable proximity to a dock, processing plant, or remote site used to house the employers and employees (Novotny et al. 1996).

When fish are close to harvest, stocking densities are kept relatively low within a netpen population of Chinook salmon, as compared to intensive production of Atlantic salmon in

Norway which can reach densities of 40 kg/m3. Chinook salmon along the western coast of

Canada are extensively reared at 4 kg/m3 (Munro 1990).

7 Ideal sites for marine cages vary from region to region and depend upon the species

selected. Since Chinook salmon’s lower seawater temperature and upper limits are about -0.7°C and 21 -23°C, areas that have not been known to drop to less than 1 -2°C would be considered optimal (Rosenthal, Scarratt & Mclnemey-Northcott 1995; Novotny et al. 1996). Under conditions of prolonged exposure to anything less than optimal could result in “superchiU”,

crystallizing the blood and stopping the heart (Saunders 1987). Fish growth and overall annual productivity are at their optimum in areas where winter temperatures remain relatively high and

summer temperatures remain on the lower end of the scale (Novotny et al. 1996).

Seawater cage culture is exposed to many other environmental and other hazards that may include algal blooms, auto-contamination, storm, or ice damage, net fouling by marine growths, predator attacks, boats colliding with cages and stress (Munro 1990).

Globally, Chinook salmon are farmed in Japan, Canada, United States, Chile and New

Zealand (Munro 1990). One of the major producing provinces in Canada is British Columbia, as

Pacific salmon are farmed on the coast of Vancouver Island in three principal regions known as

North Island (Port Hardy, Port McNeil), Central Island (Campbell River, Powell River &

Sechelt), and West Coast (Clayoquot Sound, Kyuquot Sound & Quatsino Sound) (Roth 1996).

Controversy surrounding the netpen culture of salmon has recently increased, as opponents condemn the aesthetics and negative environmental effects of this culture system

(Broussard 1991). This method of aquaculture may allow for the exacerbation of certain diseases as water exchanges are uncontrolled, leaving the netpen vulnerable to the introduction of pathogens, pollution, and noxious algae (Kent 1998a). The occurrence of disease within a netpen may be minimized by good site selection, lowered stocking densities, and careful handling of stocks (Boydstun & Hopelain 1977).

8 1.2 Disease in Netpens

Infectious fish diseases have been globally acknowledged as one of the most severe threats and constraints to the commercial success of aquaculture (Southgate 1993; Muir 2005;

Subasinghe 2005). The presence of disease in a stock of fish is typically manifested by behavioral changes and or body function throughout the entire stock, leading to a reduction in performance and even death. These changes can be due to any physical, chemical or biological effects adversely impacting physiology of the fish in an induced or natural manner (Billard 1990).

The potential effects of disease can be economically disastrous and can force a farm out of business because of poor husbandry practices (Bruno & Ellis 1996). During severe disease outbreaks stock losses of 20-80% can occur and if left untreated, complete mortality may result

(Hill 1992; Muir 2005). Trout farms in Europe have been known to have losses in the amount of

71 million dollars annually due to viral haemorrhagic septicaemia (VHS) (Hill 1992).

Natural fish populations may act as a potential reservoir of disease and parasites with the increased likelihood of disease transmission through introducing new cultured stocks (Chua et al.

2002). A review of sea louse control in Scotland highlighted that Atlantic salmon (Salmo salar) smolts free of lice were infested shortly after introduction to a marine netpen and hypothesized that migrating infected wild Atlantic salmon and feral sea trout (Salmo trutta) were the source

(Rae 2002). Additionally, high stocking densities and stressful conditions within a marine netpen housing salmonids have been known to facilitate the spread and exacerbation of disease (Kent

1998a,b; Davidson 1999; Kent & Poppe 2002). Many important diseases seen in netpens often originate in fresh water, such as furunculosis or bacterial kidney disease; in some instances, the best method of control involves the identification and removal of fish with subclinical infections while being held in freshwater, thereby limiting the introduction of pathogens into seawater netpens (Kent 1998a).

9 1.2.1 Diseases of Chinook Salmon

Many diseases of Chinook salmon can negatively impact the efficient netpen culture of this species. Causative agents can be bacterial, fungal, viral, or parasitic (Southgate 1993;

Pennell et al. 1996a; Evelyn, Kent, Poppe & Bustos 1998; Kent et al. 1998a; Kent & Poppe

2002).

The most significant bacterial cause of mortality in netpen reared Chinook salmon is

Bacterial Kidney Disease caused by a non-motile, gram positive bacterium(Renibacterium salmoninarum), affecting inflammatory cells (frequently macrophages) (Evelyn et al. 1998).

Affected fish appear dark and lethargic, often displaying clinical signs; exophthalmia, small lesions on their flanks and distended abdomens (Southgate 1993; Evelyn et al. 1998). Internally, there is chronic inflammation in the kidney, visceral organs, eyes, brain and muscle, with small white lesions on the kidney, liver, spleen and heart. Mortality can be associated with anemia

(Southgate 1993; Evelyn et al. 1998).

Fungi are also a causative agent of mortality in the netpen culture of Chinook salmon. Of particular concern is the “Rosette Agent”, an unclassified intracellular protistan parasite, as it has been known to cause up to 90% mortality in cultured Chinook (Kent et al. 1998b). Clinical signs include swollen kidneys and spleen, with marked lymphocytosis (Kerk, Gee, Standish,

Wainwright, Drum, Elston & Sogin 1995). Epizootics have only been observed in Chinook salmon during their second summer in sea water, with mortalities at their highest dining the summer and fall (Harrell, Elston, Scott & Wilkinson 1986).

Infectious hematopoietic necrosis virus (IHNV) is one of the most costly viral diseases of cultured Chinook salmon, with horizontal transmission readily occurring in both freshwater and seawater (Wolf 1988; Traxler & Roome 1993). In addition to infection and necrosis of the hematopoietic tissues (kidney, spleen, and intestinal submucosa), the virus has been found at high

10 levels in the mucus and feces of infected fish, making avoidance of infection in a netpen very difficult (Traxler, Kent & Poppe 1998; Kent et al. 2002). Other factors contributing to the extent of losses due to IHNV are increased stocking density, environmental stressors and viral latency

(Traxler etal. 1998).

Many parasites can infect Chinook salmon, and many can cause severe damage to the host. They range from crustacean parasites to protistan parasites which can induce subclinical to severe infections. Sea lice, (Lepeophtherius salmonis) are marine ectoparasitic copepods, responsible for serious disease outbreaks and high economic losses to salmon farmers (Johnson

1998; Kent et al. 2002), with clinical signs including attached sea lice, fin damage, dark colouration, skin erosion and hemorrhage. Mortality is associated with stress and the inability to maintain physiological homeostasis due to osmotic stress, anemia and hypoproteinemia (Johnson

1998; Kent etal. 2002).

Of emerging importance in the aquaculture industry is the cause and effect of protistan parasites on marine cultured Chinook salmon. Protistans consist of amoeba, flagellates, ciliates and microsporidians (Kent 1998b). Of major concern for Chinook salmon farmers are the microporidians that includeNucleospora (Enterocytozoon) salmonis and Loma salmonae (Kent

1998b).

1.2.2 Protistan Diseases of Salmon in Seawater

Protista are a diverse group of unicellular eukaryotic organisms that rely on a monogenetic life cycle. Most are classified into several phyla that include: Opisthokonta,

Amoebozoa, Excavata, Rhizaria, Archaeplastida and Chromalveolata (Adi, Simpson, Farmer,

Anderson, Anderson, Barta, Brugerolle, Fensome, Fredericq, James, Karpov, Kugrens, Krug,

Lane, Lewis, Lodge, Lynn, Mann, McCourt, Mendoza, Moestrup, Mozley-Standridge, Nerad,

Shearer, Smirnov, Spiege & Taylor 2005).

11 Some of the chief protistans affecting pen reared salmonids worldwide include

Ichthyobodo necatrix (Costia), Neoparamoeba pemaquidensis (Gill Amoebiasis), Kudoa thyrsites, L. salmonae and Hexamita salmonis (Bruno et al. 1996; Kent 1998b), all of which are either ectoparasitic or endoparasitic (Southgate 1993) thereby capable of multiplying on fish integument, within viscera or the skeletal system (Bruno et al. 1996). There are many examples of differences in the host-pathogen relationships, for example whereasK. thyrsites, a myxozoan, is known to infect a wide variety of fish hosts, particularly pen-reared Atlantic salmon and coho salmon, it does not appear to be a significant problem for chinook salmon which appear resistant to infection (Kent 2000). Generally not known to cause morbidity, this parasitic infection causes unsightly cysts and soft texture in fillets post-mortem (Moran, Whitaker & Kent 1999). In contrast, L. salmonae, a species of microsporidian, has a different range of host specificity, as farm-reared chinook and coho salmon are highly susceptible to infection, while Atlantic salmon demonstrate resistance according to preliminary reports (Hauck 1984; Shaw, Kent, Brown,

Whipps & Adamson 2000).

Feral and farmed fish living in optimal conditions have little to no burden with protistan infections, as there are few reports of heavy stock losses on farms practicing good husbandry techniques, but these pathogens have been known to rapidly increase in number in netpens when fish are stressed due to crowding and poor water quality (Bruno et al. 1996). Various other environmental changes such as currents, feral salmon migration routes, and aquatic fouling from farms in close proximities also play a role in the spread of disease (Bruno et al. 1996).

Depending on the parasite involved, treatments include the use of commercial therapeutics, formalin, fumagillin and freshwater baths, improved husbandry and antibiotic top- coated feeds (Kent et al. 2002; Kent 1998b; Southgate 1993; Bruno et al. 1996). Some protistans are exceptionally difficult to control or eradicate and may even have no known treatment, such is the case with most species of microsporidians.

12 1.3 Microsporidian Parasites

Microsporidians are eukaryotic, spore forming, obligate, intracellular protistan parasites that have no active stage outside of their host cells (Weiss 2001; Franzen 2005). As reviewed by

Weiss and Vossbrinck (1998), NSgeli was the first to recognize microsporidia, by discovering an infection in silk worms ( Nosema bombycis) in 1857. Since Nageli’s time many more microsporidia have been discovered and are now known to infect all major invertebrate and vertebrate animal groups (Wittner 1999; Weiss 2001; Mathis 2000). With the number of species exceeding 1200, in 143 genera, microsporidians are causative agents of economically important diseases in insects (silk worm, honey bees), fish and mammals (rabbits, carnivores) (Wittner

1999; Weiss 2001; Mathis 2000; Keeling & Fast 2002) and to a lesser extent have been shown to

infect birds (pigeons, chickens, parrots) and reptiles (lizards) (Lobo, Xiao, Cama, Magalhaes,

Antunes & Matos 2006; Jacobsen, Green, Undeen, Cranfield & Vaughn 1998). More recently, microsporidians in the generaEncephalitozoon, Enterocytozoon, Nosema, Vittaforma and

Pleistophora have been recognized as causing infection in humans, particularly those suffering

from immunosuppression (Wittner 1999; Mathis 2000; Weiss 2001; Keeling et al. 2002; Didier,

Stovall, Green, Brindley, Sestak & Didier 2004).

Although characterized as true eukaryotes by possessing a typical eukaryotic nucleus, a

well-developed internal membrane system and a cytoskeleton, microsporidians also exhibit many

characteristics reflective of prokaryotes (Vossbrinck, Maddox, Friedman, Debrunner-Vossbrinck

& Woese 1987; Canning 1988; Weiss et al. 1998; Mathis 2000; Franzen 2005). These features

include their small genome size (i.e. 2.3 Mb for Encephalitozoon intestinalis), ribosomes

analogous to those of prokaryotes and their lack of either peroxisomes or a typical Golgi

apparatus (Canning 1988; Weiss et al. 1998; Vavra & Larsson 1999; Mathis 2000; Weiss 2001;

Keeling et al. 2002; Didier et al 2004). Additionally, microsporidians are amitochondrial but have been shown to possess a heat shock protein (HSP70) considered a molecular signature of

13 mitochondria (Germot, Phillipe, Le Guyader 1997). It has been hypothesized that they may have possessed mitochondria in the past and lost them during the process of evolving to a highly specialized intracellular parasite (Vavra et al. 1999).

The microsporidian spore (Figure 1) wall is composed of three layers, beginning with an electron dense exospore composed of glycoprotein, an electron-lucent endospore composed of chitin and an inner plasma membrane (Walker &.Hinsch 1975; Canning & Lom 1986; Didier et al. 2004; Franzen 2005). Spore shape in most species is oval or pyriform, although rod-like, spherical and various other shapes are not unusual (Vavra et al. 1999).

Anchoring Disk

Cell Membrane Endospore Exospore Polar Tube

Nucleus

Sporoplasm

Posterior Vacuole

Figure 1 Generalized diagrammatic representation of a microsporidian spore with contents: anchoring disc, polar tube, nucleus, sporoplasm, cell membrane, endospore, exospore and posterior vacuole.

14 13.1 Microsporidian Life Cycle

Microsporidians are best known for their highly resistant spores and the possession of a unique extrusion apparatus, within the spore, consisting of a coiled polar tube (Fig. 1) attached to an anchoring disk at the apical part of the spore (Walker & Hinsch 1975; Weidner 1976; Vdvra et al. 1999; Franzen 2005). The majority of microsporidian infections are thought to occur via

ingestion of spores (Canning et al. 1986; Didier et al. 2004; Lorn & Dykova 2005), although experimental studies have successfully transmitted the parasite intraperitoneally, intramuscularly,

intravascularly or by anal gavage (Speare 1998a,b; Shaw & Kent 1999). These latter routes,

although unlikely to be used during initial contact between parasite and host, might be relevant when we consider autoinfection as might occur when a spore-filled cysts raptures within an

infected host.

Upon ingestion the spore is subjected to an environmental trigger (Keeling & Fast 2002).

Although poorly understoodin vivo, experimental studiesin vitro have demonstrated that spore germination may occur via alteration in pH, dehydration followed by rehydration, exposure to

UV light, peroxides or hyperosmotic conditions (Pleshinger & Weidner 1985; Weiss 2001;

Keeling et al. 2002). Cali & Takvorian (1999) suggest that it is a single trigger or a combination of stimuli that allow for a sequence of events to occur that result in polar tube discharge. It is hypothesized that in an acidic environment when potassium and/or sodium ions are present along with chloride ions, they trigger an increase in spore calcium concentration (Pleshinger et al.

1985). The calcium flux activates the enzyme trehalase that cleaves intrasporal disaccharide trehalose into smaller molecules (Undeen & Vander Meer 1994, 1999). As internal spore pressure increases as well as the spore’s concentration of soluble molecules (Keeling et al. 2002), transmembrane pathways open, allowing for the entry of water that reacts with the molecules to dramatically increase osmotic pressure (Cali et al. 1999). The increased intrasporal pressure is seen with significant swelling of the polaroplasts and the posterior vacuole and subsequent

15 breakdown of sporoplasmic membranes leading to the rupture of the anchoring disk and discharge of the polar tube (Weidner 1982; Weiss 2001; Keeling et al. 2002; Franzen 2005).

Measuring 50-500 pm in length and 0.1 -0.2 pm in diameter (Keohane & Weiss 1999;

Keeling et al. 2002), the polar tube is everted with such force that it pierces a host cell membrane,

inoculating the target cell’s cytoplasm with infective sporoplasm along with its nucleus (Keeling

et al. 2002; Franzen 2005). Occurring in just under two seconds (Weiss 2001), with discharge

velocities exceeding lOOpm/s, the microsporidian parasite has successfully invaded its target cell while avoiding the host cell plasmalemma, thereby preventing interaction with some of the host’s

signaling mechanisms (Cali et al. 1999) and infecting the new cell without being recognized as an

invader (Keeling et al. 2002; Keohane et al. 1999).

Successful invasion marks the beginning of the proliferative phase (Cali et al. 1999)

which may ensue in different tissues far from the site of spore germination (Canning et al. 1986).

Wandering cells, such as macrophages, monocytes and/or undifferentiated mesenchyme cells are believed to transport the parasite from the lamina propria (Canning et. al 1986; Lorn et al. 2005),

while participation of intraepithelial leucocytes that naturally occur in the fish gut (Bernard, Six,

Rigottier-Gois, Messiaen, Chilmonczyk, Quillet, Boudinot & Benmansour 2006) have also been

suggested as transport cells due to their ability to migrate to interstitial tissues and blood

(Rodriguez-Tovar, Wright, Wadowska, Speare & Markham 2002). Rodriguez-Tovar et al. (2002) proposed under an “Internalization Hypothesis” that some pillar cells of the gills act as phagocytes to ingest infected “wandering cells”, at which time the secondary-infected pillar cells disrupt connections with neighbouring cells, triggering the uninfected adjacent pillar cells to retract and rearrange their flanges to isolate the infected cell from the blood.

Cell growth and proliferation follows and is known as merogony (or schizogony) and depending upon the species of microsporidian; this phase is either initiated upon infection or within the final target cell (Canning et al. 1986; Cali et al. 1999) and is characterized by asexual production (binary or multiple fission) of numerous parasitic stages within the invaded host cell

16 known as meronts (or trophozoites) (Canning et al. 1986; Keeling et al. 2002; Lom et al. 200S).

During infection with L. salmonae, Rodriguez-Tovar, Wright, Wadowska, Speare and Markham

(2003) noted that merogony takes place within transport cells, specifically in macrophages, while

PCR techniques and in situ hybridization have detected the dividing parasite’s DNA within the

heart as early as 2 days post exposure to infective material (Sanchez, Speare & Markham 2000;

Sanchez, Speare, Markham, Wright & Kibenge 2001).

Following merogony, the life cycle of microsporidians enters sporogony, (thought to

occur exclusively upon entry into pillar cells) at which time presporal cells, denoted sporonts, are

initially produced (Canning et al. 1986; Sanchez et al. 2000; Keeling et al. 2002). Sporonts

divide presumably by binary fission into sporoblasts (presporal cells), with production ranging

from bisporous to multisporous, depending on the species of microsporidian (Keeling et al. 2002;

Franzen 2005). These presporal cells undergo a period of morphogenesis, involving the

production of a proteinaceous exospore and a thicker chitinous endospore surrounding the

organelles of what is now considered a true spore (Canning et al. 1986; Cali et al. 1999; Keeling

et al. 2002).

The invaded host cell enlarges and its surface becomes modified either by producing

microvillus-like structures or by mass formation of pinocytotic vesicles, to accommodate further

nutrient absorption from surrounding tissues required for the developing parasite, as seen in

G/ngea-induced xenomas in winter flounder, Pseudopleuronectes( americanus) (Weidner 1976;

Lom et al. 2005). Depending on the species of microsporidian, the host cell and its nucleus may undergo severe hypertrophy (Lom & Nilsen 2003) and in the early stages of host cell enlargement, the developing parasite is either distributed uniformly throughout the host cell cytoplasm,Loma ( spp.) or stratified by developmental stagesGlugea ( spp.) (Shaw et al. 1999;

Lom et al. 2003). Some developing parasites Encephalitozoon( spp.) may be surrounded by a membrane of host cell origin known as the parasitophorous vacuole or a sporophorous vesicle

(Glugea spp.) of parasitic origin (Magaud, Achbarou & Desportes-Livage 1997; Lom et al. 2003).

17 As reviewed by Sprague and Hussey (1980), maturation is exemplified by the parasites occupying

the centre of the invaded cell with the host responding by laying down a layer of connective

tissue surrounding the recently enlarged cell, now coined a “xenoma”. A fully developed xenoma

is completely filled with numerous mature spores, with very little host cell components remaining

(Canning et al. 1986; Shaw et al. 1999). Within the xenoma, the parasite and host cell represent a

symbiotic relationship (Weissenberg 1968), as the parasite benefits by having a suitable

environment for proliferation, while avoiding host recognition and the host benefits by reducing

the spread of infection by confining the parasite to one cell (Sprague et al. 1980; Canning et al.

1986; Lom et al. 2005). In some microsporidian genera, such as Pleistophora, the developmental

stages are not confined to xenomas and are located within the host cell cytoplasm and have the

ability to develop diffusely in various tissues of their host such as muscle fibers. (Dykova & Lom

1980; Canning et al. 1986; Lom et al. 2003). Such microsporidia are termed pansporoblastic

(Weiss et al. 1998).

1 J.2 Microsporidians in Human and Animal Health

Likely to have gone undiagnosed for over a hundred years, as the initial documented

discovery of microsporidiosis of animals was in 1857, the first reported case of microsporidiosis

in humans occurred in 1985 (Desportes, Le Charpentier, Galian, Bernard, Cochand-Priollet,

Lavergne, Ravisse & Modigliani 1985; Weiss & Keohane 1999). The diagnosis came two years

after the identification of HIV as the causative agent of AIDS, when the microsporidia

Enterocytozoon bieneusi was discovered in fecal samples of HIV-infected patients (Desportes et

al. 1985). Since then, many genera of microsporidians includingEncephalitozoon, Nosema,

Brachiola, Trachipleistophora, Pleistophora, Microsporidium, and Vittaforma that were previously known to infect insects, fish, reptiles, rodents, rabbits, fur bearing animals and primates have been shown to infect humans (Kotler & Orenstein 1999). Of all human causes of microsporidiosis,E. bieneusi and Encephalitozoon cuniculi are the most commonly reported

(Didier 1998) and depending on the host’s immune status, these species can cause acute to

18 chronic disease (Didier 1998; Didier et al. 2004; Didier 2005). Infection of immune-competent hosts is mainly accompanied by an acute, self-limiting diarrhea which persists for 2-3 weeks

(Bryan, Weber & Schwartz 1997; Okhuysen 2001; Muller, Bialek, Kamper, Fatkenheuer,

Salzberger & Franzen 2001), whereas infection of immune-deficient hosts, including AIDS patients with less than one hundred CD4+ T-cells/ml blood manifests in severe persistent diarrhea, fever, loss of appetite, weight loss and subsequent wasting disease (Orenstein 1991; Asmuth,

Degirolami, Federman, Ezratty, Pleskow, Desai & Wanke 1994; Kotler & Orenstein 1999;

Okhuysen 2001)

E. cuniculi has had a major impact on animal heath, causing heavy losses in blue foxes in

Northern Europe and high morbidity in captive monkeys (Didier et al. 1998; Snowden &

Shadduck 1999; Deplazes, Mathis & Weber 2000), presumably caused by the infection initiating hyperimmune responses resulting in the formation of immune complexes and subsequent renal failure (Mohn & Nordstoga 1975; Shadduck & Orenstein 1993; Snowden, Didier, Orenstein &

Shadduck 1998). E. cuniculi has also been associated with ascites in experimentally infected mice and motor paralysis, torticollis and convulsions in naturally and experimentally infected rabbits (Pattison, Clegg & Ducan 1971; Snowden et al. 1999; Didier 2005) and vestibular disease in infected rabbits (Pattison et al. 1971; Snowden et al. 1999; Kiinzer, Gruber, Tichy, Edelhofer,

Nell, Hassan, Leschnik, Thalhammer & Joachim 2008).

Transmission of microsporidians in humans is strictly horizontal however there are recorded cases of vertical transmission in rodents, rabbits, carnivores and non-human primates

(Snowden et al. 1999). Human microsporidiosis predilection sites are generally along the gastrointestinal and respiratory tracts (Didier 1998; Didier et al. 2004; Didier 2005), therefore transmission routes are thought to be fecal-oral, oral-oral, and inhalation of contaminated aerosols or ingestion of contaminated food or water (Bryan & Schwartz 1999; Mota, Rauch & Edberg

2000; Weber, Deplazes & Schwartz 2000; Deplazes et al. 2000).

19 As reported by Lom et al. (2003) fish are hosts to 156 species of microsporidia. Several of these are held responsible for declines of entire commercial fisheries (Ralphs & Matthews

1986) and they are known to parasitize fish held in both marine and freshwater (Weissenberg

1968; Kent, Elliot, Groff & Hedrick 1989). In 1954, Glugea hertwigi was implicated in the declines of rainbow smelt ( Osmerus mordax), fishery of New Hampshire (Haley 1954). Eels

(Anguilla japonica), reared in Korea are typically housed in heating-house systems that maintain water temperatures between 25-30°C and when infected with the non-xenoma forming microsporidia, Heterosporis anguillarum have been demonstrated to have high transmission potential and morbidity and mortalities associated with lesions and scattered spores within muscle fibers (Joh, Kwon, Kim, Kim, Kwon, Park, Kwon & Kim 2007). Additionally, the microsporidium,Enterocytozoon salmonis has in the past been responsible for severe anemia in

Chinook salmon in Washington by means of infecting hemoblastic cells of the spleen and kidney

(Elston, Kent & Harrell 1987). Also known as plasmacytoid leukemia, E. salmonis is thought to be the causative agent responsible for variable levels of mortality, with one farm reporting losses up to 50% of fish in their second year at sea (Kent, Eaton & Casey 1997). Other threats to commercial aquaculture caused by fish microsporidiosis include presence of large cysts, liquefaction of the muscles (Pulsford & Matthews 1991), starvation causing a decrease in growth rates, reduced swimming speed leading to increase in predation (Hauck 1984; Matthews &

Matthews 1980; Sprengel & Luchtenberg 1991). Additionally, fecundity of smelts infected with the microsporidian parasiteGlugea hertwigi, by means of mechanical interference and complete occlusion of the vents, thereby inhibiting the discharge of milt and eggs (Haley 1953; Sindermann

1963).

1.4 Loma salmonae

Associated with xenoma formation (Shaw et al. 1999), the genusLoma contains nine species known to infect commercially important stocks (Shaw et al. 1999), particularly salmonids. Morbidity and mortality due to severe tissue damage has been documented in

20 rainbow trout ( Oncorhynchus mykiss), brook trout (S. fontinalis), Coho (O. kisutch) and Chinook salmon (O. tshawytcha) with L. salmonae being identified as the causal agent (Kent et al. 1989;

Kent, Dawe & Speare 1995). Kent et al. (1989) reportedL. salmonae-associated losses of pen- reared Coho salmon periodically exceeded 30% annually. Severe infections with the parasite have been well documented in Pacific salmon and rainbow trout in the Pacific Northwest (Hauck

1984; Kent et al. 1989), Eastern United States (Markey, Blazer, Ewing & Kocan 1994) and

Scotland (Bruno et al. 1995).

1.4.1 Transmission

Once considered a freshwater parasite simply maintaining pathogenicity upon transfer to seawater (Kent et al. 1989), L. salmonae has now been characterized as capable of transmitting infection in both freshwater and marine environments (Kent et al. 1995; Shaw et al. 1998). Much like other microsporidians, L. salmonae has been shown to have various routes of transmission.

When spores are liberated from a raptured xenoma, they may be taken into or delivered to an individual in various ways to cause infection. In an interesting laboratory study by Shaw, Kent and Adamson (1998), Pacific salmon ( Oncorhynchus spp.), were subjected to various methods of infection and demonstrated that delivery of infective spores via anal gavage,per os, cohabitation with infected tank mates and intramuscular, intraperitoneal and intravascular injections were successful at transmitting the parasite. However, direct application of spores to the gill was unable to produce infection, disproving Hauck’s (1984) theory that pillar cells phagocytized parasites present in water that passed over the gills (Shaw et al. 1998). Additionally, the results indicate that regardless of the route of delivery, the development ofL. salmonae has predilection for the gills (Shaw et al. 1998). Although the laboratory findings are useful in a controlled setting, transmission via per os and cohabitation are the most likely of scenarios occurring in marine netpens.

A study of the coastal waters of British Columbia by Kent, Traxler, Kieser, Richard,

Dawe, Shaw, Prosperi-Porta, Ketcheson, and Evelyn (1998) found that wild ocean-caught

21 salmonids (Chinook salmon, chum salmon Oncorhynchus keta, Coho salmon O. kisutch, sockeye salmon O. nerka, and pink salmon O. gorbuscha), had ongoing L. salmonae infections, and therefore these fish could be potential reservoirs of infection. Since fish-to-fish transmission has been demonstrated as the mode of infection (Shaw et al. 1998), particular attention should be given to the potential threat feral fish pose to farmed fish.

1.4.2 Diagnosis and Clinical Signs

L. salmonae is typically identified in fish by microscopic examinations of intact or ruptured xenomas and spores within gill filaments (Hauck 1984; Weber, Schwartz & Deplazes

1999) (Fig. 2 & 3). Wet-mount preparations of moderately to heavily infected gills can also easily detect xenomas under low-level microscopy, while a higher magnification reveals densely packed spores within (Kent et al. 2002). Advanced techniques are also available for early detection of L. salmonae by means of a sensitive polymerase chain reaction (PCR) test using the parasite’s rDNA sequence (Docker, Devlin, Richard, Khattra & Kent 1997).

Clinical signs of infection vary in intensity depending on the species infected (Speare,

Arsenault & Buote 1998) and appear upon the rupture of the xenomas within the gill lamellae

(Canning et al. 1984; Hauck 1984). They are manifested as severe respiratory distress and lethargy (Kent et al. 2002), as a result of lamellar fusion due to high levels of epithelial hyperplasia (Canning et al. 1984) (Fig. 4). Additionally, Hauck (1984) noted heavy infections in

Chinook salmon resulted in the occlusion of arteries, darkening of the tail, significant necrosis of cartilage and musculature of the tail and head, pericarditis of the bulbus arteriosus and hyperplasia of the heart tissues. Additionally, severe branchial inflammation is noted upon rupture of xenomas and is thought to be associated with the host-reaction targeting the chitin-rich wall of spores remaining within gill tissues (Speare, Brackett & Ferguson 1989; Kent et al. 1995;

Vavra & Larsson 1999). Severely infected Chinook salmon demonstrate unpaired swimming, reduced growth rates and increased mortality due to predation and starvation (Hauck 1984;

Speare, Daley, Markham, Sheppard, Beaman & Sdnchez 1998).

22 Fig. 2 Histological section of rainbow trout gill infected with Loma salmonae. Shown are four large parasitic xenomas containing many spores. H&E. Scale bar = 50 pm

23 Fig. 3 Histological section showing a rupturedLoma salmonae xenoma in the gills of an infected rainbow trout. (Typical example shown by arrowhead). H&E. Scale bar = 50 pm

24 Fig. 4 Histological section of a Loma salmonae-infected rainbow trout gill showing epithelial hyperplasia. Lamellae show several thickened areas of epithelial tissue. H&E. Scale bar = 50 pm

25 1.4 J Immunological Resistance

Laboratory models of infection in various salmonids have demonstrated levels of induced resistance to re-infection with L. salmonae. Strong protection againstL. salmonae has been demonstrated in both rainbow trout and Chinook salmon undergoing a secondary per os exposure to infected gill material. After recovery and subsequent re-challenge both species were 100% protected against disease as measured by lack of xenoma formation at optimal temperatures for the parasite (Speare, Beaman, Jones, Markham & Arsenault 199S; Kent et al. 1999).

Additionally, rainbow trout infected outside the optimal temperature range for xenoma formation

(9-20°C) displayed protective responses to re-infection when re-challenged within the permissive temperature for xenoma formation (Beaman, Speare, Brimacombe & Daley 1999). These findings are significant for the fact that hatchery stock orally exposed to the parasite below the permissive temperature range could mount protective responses upon re-exposure to the parasite within marine netpens whereL. salmonae is endemic, all without running the risk of developing the disease.

Further resistance studies include rainbow trout that received intraperitoneal injections of a fresh spore preparation. These fish also demonstrated complete resistance against branchial xenoma formation when orally re-challenged (Speare et al. 1998b). Although not without commercial precedence, the use of viable pathogens to evoke immunity is relatively controversial, therefore, the study also included a freeze-thaw preparation ofL. salmonae spores

(considered to be rendered relatively non-viable), that when injected intraperitoneally was capable of inducing protective responses in just over half of the fish immunized upon oral re­ exposure. Although the fresh spore preparations were able to confer favourable results, the results indicated that the initial IP exposure to fresh spores allowed for 59% of the population to develop xenomas while only 4.7% displayed xenoma when IP injected with freeze-thaw preparations (Speare et al. 1998b). Interestingly, this is the first study to demonstrate that freeze-

26 thawing (inactivating) spores ofL. salmonae is able to render the parasite significantly less virulent to naive fish than those being delivered unaltered.

The discovery of two variants ofL. salmonae (exact taxonomic relationship pending) has

led to further resistance studies in rainbow trout. Sanchez, Speare, Markham & Jones (2001b)

isolated and amplified an Oncorhynchus-associated (OA) and Salvelinus-variant (SV) strains of

L. salmonae, each haying varying affinities for rainbow and brook trout, the latter having low virulence in rainbow trout and high virulence in brook trout (Sanchez et al. 2001b). It is with this

information that Sanchez et al. (2001c) evaluated the ability of the low virulence variant in rainbow trout (SV) delivered per os to confer protection against re-exposure methods toL. salmonae OA strain. It was determined that although the Salvelinus-variant has low affinity to rainbow trout it is able to confer levels of protection against re-exposure to the high virulence

Oncorhynchus-associated L. salmonae. Additionally, whole-spore vaccine preparations of the

Salvelinus-variant delivered intraperitoneally have also been shown to boost immune responses

against re-infection to the high virulent OA variant in rainbow trout (Speare, Markham & Guselle

2007). Since fish entering the marine netpen are at the highest risk for infection, the inoculation

of hatchery stock would be the most likely application, and therefore the freeze-thaw preparation would be the safest approach as there little to no risk of a farmer infecting their nai've stocks and risking increased levels of morbidity and mortality.

Experimental host specificity L.of salmonae is not limited to Oncorhynchus spp. but extends also to theSalvelinus and Salmo genera, as brook trout ( Salvelinus fontinalis) and brown trout (Salmo trutta) can be infected with the microsporidian (Shaw, Kent, Brown,Whipps &

Adamson 2000). Unlike members of the Oncorhynchus genus, experimental studies using brook trout and the high virulent SV variant demonstrated that brook trout do not mount a protective response against oral re-infection withL. salmonae SV. Research involving other species of the genus Salvelinus is required before any real conclusions can be made concerning the lack of immunity against re-infection toL. salmonae SV.

27 Compared to bacterial and viral pathogens, the immune response in fish to microsporidial

infections has in the past been relatively undefined (Shaw et al. 1999), although recent studies have further characterized the involvement or lack thereof of immune defense lines against

microsporidia (Rodriguez- Tovar, Markham, Speare & Sheppard 2006b). Some initial

experimental studies on the immune responses toL. salmonae have deduced that resistance is cell mediated in rainbow trout. Passive immunization of naive fish with sera from previously infected rainbow trout was unable to protect the fish from subsequent infection when challenged (Sanchez,

Speare & Markham 2001a), dismissing the humoral response as being protective. Conversely, a marked lymphocyte proliferation, involving head kidney mononuclear cells (MNC), in response to antigen of L. salmonae in exposed and protected fish was described (Rodriguez-Tovar et al.

2006b). Furthermore, with other microsporidia, it has been recognized that a form of T cell reactivity is required for cellular defense and the proliferative response noted in previously exposed fish toL. salmonae could implicate T cell activity in this resistance, thereby supporting the involvement of cellular immunity (Rodriguez-Tovar et al. 2006b).

The use of adjuvants to boost the immune system is a regular practice in the medical world, and has been demonstrated to be weakly effective on its own and significantly effective when mixed with antigens (Anderson 1997). Much like adjuvants, immuno-stimulants are also known to boost immune responses and in experimental studies by Guselle, Markham & Speare

(2006 & 2007), the protective effects of intraperitoneal injections with the immuno-stimulant

Pro Vale™, a p-1,3/1,6 glucan (Stirling Products North America Inc., Charlottetown, Prince

Edward Island, Canada), effectively reduced the xenoma intensity within the gills by 91% when the fish were injected 3 weeks post exposure toL. salmonae. Compared to chemotherapeutants experimentally used to controlL. salmonae, the level of protection conferred via injection with fJ-

1,3/1,6 glucans is much greater (Guselle et al. 2006 & 2007; Speare et al. 2007). Unlike oral administration, intraperitoneal administrations of immuno-stimulants are not as feasible in large hatcheries or netpen farming environments due to the quantity of fish, the cost and stress

28 associated with handling (Scott 1993; Bruno et al. 1996) even though they tend to generate higher

levels of protection against disease. It is believed that immuno-stimulants allow for disease resistance by increasing lysozyme and complement activities and activating macrophages (Scott

1993; Cook, Hayball, Hutchinson, Nowak & Hayball 2001).

The humoral response in fish is described as the first line of immune defense (Alvarez-

Pellitero 2008), that targets bacteria by synthesizing and releasing specific antibodies into the bloodstream, particularly those of the IgM isotype (Cushing 1970; Scott 1993; Watts, Munday &

Burke 2001). These antibodies react and bind to particular antigens associated with bacteria, typically leading to engulfinent by macrophages and destruction of the antigen (Barton 1996).

The second line of defense is an adaptive response, involving cell-mediated immunity and differs

from a humoral response by focusing on viruses, fungi, and mycobacteria and is characterized by the direct immunological effects the cells of the immune system (B and T cells) have on the

foreign body (Barton 1996). After receiving stimulus from B cells, T cells differentiate into

subpopulations depending on the type of signal received, they may be directly cytotoxic, induce

an inflammatory response, aid in the assembly of antibodies, regulate or inhibit other immune

responses and/or produce specialized proteins known as interleukins to contribute to the overall effort to rid the body of antigen (Sell 1987; Ellis 1988; Barton 1996). Although immunological resistance to L. salmonae in salmonids has been studied extensively, other avenues have been analyzed in mounting protective responses against MGDS.

1.4.4 Drug Treatment ofL. salmonae infections

Chemotherapeutants, particularly antibiotics, administered in feed have been used by fish farmers for some time to treat bacterial disease in hatcheries and marine netpens (Weston,

Phillips & Kelly 1996). However, there are currently no pharmacological agents licensed for use in controlling L. salmonae or any other microsporidian parasites affecting farmed fish (Speare et al. 1998b), but several experimental studies have been performed to evaluate various treatment regimes.

29 The antimicrobial agent fumagillin dicyclohexamine (DCH) demonstrated an ability to limit the effects of various microsporidian (Hedrick, Groff & Baxa 1991) and myxosporidian species (Molnar, Baska & Szekely 1987; Hedrick, Groff, Foley & McDowell 1988; El-Matbouli

& Hoffman 1991; Sitja-Bobadilla & Alvarez-Pellitero 1992). A natural product of the fungus

Aspergillus fumigatus (Hanson & Eble 1949) and known to inhibit RNA synthesis (Jaronski

1972), fumagillin DCH in non-lethal amounts incorporated into feed was initially regarded as a means of controlling xenoma formation in Chinook salmon (0. tshawytscha) in a study lasting only 6.S weeks (Kent & Dawe 1994). It was later determined to simply delay xenoma onset and substantially reduces xenoma burden by 10 weeks post exposure (Speare, Athanassopoulou,

Daley & Sanchez 1999). Due to the delay in xenoma onset, the study carried out by Kent et al.

(1994) did not allow sufficient time for xenoma formation and therefore it was erroneously concluded that the effects of the agent allowed for complete protection. Although currently only licensed in Canada for the treatment of microsporidiosis of honey bees (Williams, Sampson,

Shutner & Rogers 2008), the application of fumagillin DCH to retard xenoma formation could be beneficial to get infected fish through the months of summer when water would be at its warmest with lowest oxygen content. Treatment allows for formation and dissolution of xenomas occurring later in the season when water is cooling, allowing for an increase in dissolved oxygen and would lessen the resulting respiratory stress (Speare, Ritter & Schmidt 1998d).

TNP-470, an analog of fumagillin DCH delivered orally in feed was initially deemed as having the ability to limit the severity of infection withL. salmonae in Chinook salmon

(Oncorhynchus tshawystcha) (Higgins, Kent, Moran, Weiss & Dawe 1998). Much like the study by Kent et al. (1994), Higgins et al. (1998) may have misinterpreted the efficacy since the trial was terminated 8 weeks PE at which point xenoma formation may have just begun due to the potential retardation effects of the TNP-470, much like that of fumagillin DCH. Further studies are required to allow longer incubation periods, in order to deem the agent effective at controlling

MGDS or simply capable of delaying onset of disease.

30 Similar results as demonstrated with application of fumagillin DCH and TNP-470 were found as the effects of the anti-protistan quinine hydrochloride onL. salmonae were investigated by Speare et al. (1998d). Commonly used as a treatment formalaria (Felix & Danis 1987; Scott

1993; Busari & Busari 2008), and believed to interfere with parasite DNA/RNA biosynthesis

(Tracy & Webster 1996), quinine hydrochloride, when incorporated into feed, delays xenoma formation up to 30%, but unlike previously studied drug therapies, is unable to reduce the ultimate size or abundance of xenomas in affected fish (Speare et al. 1998d).

Furthermore, dietary treatments againstL. salmonae using monensin, a sodium ionophore were demonstrated to successfully delay xenoma formation and reduce xenoma burden (Speare,

Daley, Dick, Novilla & Poe 2000; Becker, Speare, Daley & Dick 2002). Specifically active on the Golgi apparatus (Dinter & Berger 1998), which is thought to be a key organelle in polar tube development (Vavra & Larsson 1999), monensin modifies intracellular ion channels (Speare et al.

2000) and glycosylates polar tube proteins, thereby inhibiting sporogony (Speare et al. 2000).

Of all dietary treatments, fumagillin DCH, albendazole and monensin were able to reduce the xenoma burden during L. salmonae infections by 57%, 67% and 85% at week 7 PE, respectively (Kent et al. 1994; Speare et al. 1999a; Becker et al. 2002). Even though dietary treatments using various compounds have demonstrated promising results, their appeal is weakened by the fact that dose rates per fish are variable, as fish may be off feed for various reasons such as sickness (Collins 1993; Lall & Tibbetts 2009), stress (Volkoff & Peter 2006; Lall et al. 2009), competition (Pennell & Mclean 1996; Lall et al. 2009) or the chemical agent incorporated into the feed may change the flavour to a less desirable one (Geurden, Cuvier,

Gondouin, Olsen, Ruohonen, Kaushik & Boujard 2005; Lall et al. 2009). With oral delivery having its limitations, an avenue worth exploring when it comes to delivering accurate dosages is vaccination (Sommerset, Krossoy, Biering & Frost 2005).

31 1.4.5 Vaccines againstL. salmonae

Although good husbandry practices and limited stress are key factors in the prophylaxis

of disease in marine netpens, the use of commercial vaccines to protect against infectious agents

in aquaculture has expanded rapidly, likely due to the emergence of bacterial resistance to various

chemotherapeutants, particularly antibiotics (Bruno et al. 1996) and the drive towards more

intensive cultures of highly-valued marine species (Sommerset et al. 2005). Although requiring

more manpower than oral treatment, vaccination, particularly into the intraperitoneal cavity of the

fish is generally regarded as the most effective site of injection (Ellis 1988; Sommerset et al.

2005). Once reported to confer protection rates greater than 60% against disease (Scott 1993),

this claim does not take into consideration the many experimental vaccines that fail to implement

any protective responses. Furthermore, many vaccines are not deemed commercially safe and

available to farmers until many trial and errors have occurred (Sommerset et al. 2005). To date,

there are effective vaccines on the market against such diseases as furunculosis, vibriosis and

enteric red mouth (ERM) (Saunders 1995; Bruno et al. 1996), but there are none currently

available against microsporidiosis in aquaculture (Speare et al. 1998b).

An experimental freeze-thaw (inactivated),L. salmonae spore-based vaccine delivered

six weeks prior to oral exposure to infected material was demonstrated to confer near complete

protection against experimental re-exposureL. to salmonae spores (Speare et al. 1998b;

Rodriguez-Tovar, Becker, Markham & Speare 2006a). With these findings, a freeze killed

(inactivated) whole-spore vaccine using the low virulence strain (SV) ofL salmonae was

investigated and when delivered intraperitoneally at six weeks prior to exposure, had the ability to

confer 95% protection against a challenge with the high virulence (OA) strain in rainbow trout

(Speare et al. 2007). These findings are extremely encouraging as vaccinating smolts 6 weeks prior to sea water transfer may allow for protective responses in a netpen where the parasite is

endemic, thereby reducing the xenoma induced pathology and mortalities as a result of infection.

32 Another beneficial aspect of vaccination is the ability of heterologous species to confer cross-protection by delivery via injection. Surprisingly, the use of differing species than the causative agent to resist disease is a relatively old practice with one of the most famous demonstrated by Edward Jenner in 1796 (Behbehani 1983). Jenner vaccinated a young boy with material taken from a cowpox lesion and after 6 weeks, the boy was challenged with smallpox and complete resistance to disease was noted (Behbehani 1983). Since Jenner’s time, many heterologous vaccines have been developed and put to the test experimentally. Recently, an experimental study using a ferretMustela ( putorius furo), animal model exhibited cross- protective effects of various strains of the highly pathogenic avian influenza virus (HPAI) of the

H5N1 subtype when vaccinated intramuscularly and challenged intratracheally (Baras, Stittelaar,

Sime, Thoolen, Mossman, Pistoor, van Amerongen, Wettendorf, Hanon & Osterhaus 2008). To date, the use of related microsporidian species for heterologous vaccination againstL. salmonae has not been experimentally studied, and its possibilities are appealing.

1.5 Research Objectives

Given the limited range of pharmacological agents with a demonstrable effect on the course of L. salmonae infections and the emerging evidence that vaccination against L. salmonae may be a viable approach, the overall objective of this thesis is to gain a better understanding of the efficacy of vaccination and/or protection provided by a prior exposure, under a range of scenarios, including an evaluation of several novel vaccine candidates. The outcome variable used to determine protection was the morphometric assessment of the number of xenomas forming on the gills of fish following exposure toL. salmonae, and this was used to answer the following questions:

1. What is the relative protection against a high dose oral exposure toL. salmonae spores, in

rainbow trout which have recovered from infections arising from a prior high dose oral

exposure or a presumed low dose, co-habitation exposure?

33 2. Can we demonstrate cross-protection? Specifically, can vaccine preparations derived

from various Glugea spp. be used to protect trout againstL. salmonae?

3. What is the relative performance of Glugea-based vaccines, compared to Loma-based

vaccines to protect against L. salmonae?

4. Can the addition of an immunostimulant improve the efficacy of naturally acquired

immunity againstL. salmonae?

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47 2. COMPARATIVE RESISTANCE TO LOMA SALMONAE IN RAINBOW TROUT (ONCORHYNCHUS MYKISS) SUBSEQUENT TO RECOVERY FROM AN INITIAL LOW DOSE COHABITATION EXPOSURE

2.1 ABSTRACT

In an effort to more fully understand the vulnerability and acquired resistance of netpen farmed populations of salmon to Microsporidial Gill Disease of Salmon (MGDS) caused byLoma salmonae, a disease in which large spore filled xenomas form within the gill vasculature, this study follows a group of 30 gram rainbow trout initially exposedL. to salmonae through a 4 week cohabitation with heavily infected donor fish, in order to determine to what extent these recipient fish could subsequently, upon recovery, resist a high dose challenge withL. salmonae. Does mild infection, as might occur when netpen fish become infected from natural reservoirs of Loma, confer resistance? Subsequent to being housed with donor fish, 100% of the recipient fish developed xenomas, and the level of infection was classified as low based on non-lethal visual assessment of their gills. Using this screening method, gills appeared to be free of xenomas by week 10 and there was no evidence of autoinfection based on the absence of newly developing small xenomas. Two weeks after xenomas had cleared from gills, these fish, along with a group of previously unexposed control trout, were challenged with an experimental single high dose oral challenge with gill tissue heavily laden with xenomas. Five weeks following this exposure the gills of these fish were assessed and whereas it was found that 100% of the control trout had xenomas on their gills, only 50% of the previously exposed fish had xenomas. This difference was significant (p<0.001). Furthermore, the previously exposed fish had 90-93% fewer xenomas per gill arch compared to the control trout (p<0.001). .The results show that, under laboratory conditions, light infections acquired through cohabitation confer significant protection against subsequent high dose exposure; the results are discussed in context of attempts to understand the severe seasonal nature of MGDS in fanned salmon.

48 2.2 INTRODUCTION

Since its recognition in 1988 (Speare et al. 1989), MGDS caused by the obligate

intracellular pathogenL. salmonae (Lom 2002), has become an endemic seasonal cause of morbidity and mortality affecting populations of Chinook salmonOncorhynchus ( tshawytscha) that are being commercially farmed along coastal British Columbia. Outbreaks most often occur in late summer and early autumn, and primarily affect salmon which have been in the ocean for over 12 months and are reaching a size and weight range that is near their harvest size and highest economic value (Kent et al. 1989). Compared to many other diseases, this vulnerability of larger well grown fish that have been in netpens for many months is unusual; for many other diseases of significance to salmon fanning, such as Infectious Pancreatic Necrosis Vims (IPNV), severe disease outbreaks are more typically found in juvenile fish and/or fish that have been recently moved from freshwater hatchery sites to seawater sites (Traxler et al. 1998; Jarp, Gjevre, Olsen &

Bruheim 1995). Although temperature is an important factor for MGDS, and summer/autumn temperatures of 13-17 °C are ideal for the parasite development, it would be anticipated that salmon would be affected during their first summer/autumn in netpens rather than their second.

Microsporidians, includingL. salmonae, are easily transmitted from fish-to-fish by several routes including cohabitation (Kent et al.1995; Shaw et al. 1998; Speare et al. 1998a;

Baxa-Antonio, Groff & Hedrick 1992). Cohabitation is a common route of transmission for many parasites not requiring intermediate hosts, such asIchthyophthirius multifiliis, Gyrodactylus salaris and Lepeophtheirus salmonis (Xu, Klesius & Panangala 2006; Lindenstrom, Collins,

Bresciani, Cunningham & Buchmann 2003; Johnson 1998). For this reason, parasitic diseases frequently emerge as significant production limiting problems in aquaculture settings where stocking densities are high, periods of cohabitation are prolonged, infected carcasses may go unnoticed, and the degree of separation of farmed stocks from wild fish is insufficient to limit pathogen ingress. Microsporidian infections in particular are easily transmitted from fish-to-fish via ingestion of infected tissues or of free spores in the environment (Kent et al. 1995), and

49 therefore cohabitation with infected individuals has the capacity to cause disease (Shaw et al.

1998; Speare et al. 1998a), due to the relative ease in which the parasite is transferred from one fish to another via cannibalism or the fact that gill tissue deteriorates rapidly after death. Any tissue fragments containing spores can be released into the surrounding environment resulting in the exposure of healthy fish to the parasite (Baxa-Antonio et al. 1992; Kent et al. 1995).

With respect toL. salmonae, potential reservoirs of infection, in native salmon stock, have been documented by Kent et al. (1998a); in their study they have shown that many species of native Oncorhynchus salmon, found in abundance in the vicinity of fish farms, carry low levels of L. salmonae within their gills. These species include Chinook (O. tshawytscha), coho (O. kisutch), pink (O. gorbuscha), chum (O. keta), sockeye(O. nerka) salmon, along with rainbow trout (O. mykiss) and 2 members of other salmonid genera including genus Salvelinus, (brook trout S. fontinalis) and genus Salmo (brown salmon S. trutta). Although the presence of these reservoirs of infection, and the ease by which infection spreads helps us understand why the disease might be endemic in farmed populations cultured in these waters, it does not help to explain why severe infections of farmed populations occur so late in the production cycle. This pattern of disease is even more challenging to understand given the ability of several salmonids, including Chinook salmon, to develop protective immunity subsequent to recovery from MGDS

(Kent et al. 1998a,b; Kent, Dawe & Speare 1999). Taken together, these findings would be more compatible with a pattern of disease in farmed stock in which the first episode would develop during the salmon’s first summer in seawater netpens when temperatures rise to those favourable to the parasite; those that recover would be expected to be resistant in their second year. This, however, is not the case.

Developing a laboratory infection model through which a better understanding of the infections acquired can be gained through cohabitation, and the subsequent host response to this type of infection, including the induced protective responses, has obvious economic practicality.

This is particularly the case for those diseases, where studies have shown that fish can mount an

50 effective protective response. Many of these results have been derived from laboratory studies,

often where the primary exposure to disease is not through cohabitation; it may be difficult to use

these studies to directly predict the degree of protection afforded by a lower dose cohabitation

exposure.

Previous laboratory work with the surrogate species rainbow trout (O. mykiss), has

demonstrated that oral introduction of L. salmonae causes the infection to develop in an identical

manner as in Chinook and Coho salmon; initial xenoma formation occurs within the gill vasculature by the fifth week after exposure and the rupturing of the spore-laden structures has been observed to occur as early as 7-10 weeks post exposure (Speare et al. 1998a). Little to no clinical signs are observed during the development of xenomas (Kent et al. 1995), although upon xenoma dissolution, severe branchial inflammation is noted (Kent et al. 1995). To a lesser extent,

laboratory studies using the target species Chinook salmon have been employed, due to their extreme levels of mortality under laboratory conditions when infected withL. salmonae and their

susceptibility to other diseases (Speare et al. 1998a). The use of rainbow trout as a surrogate has

shown that strong protective immunity develops in fish that have recovered from high dose

challenges with L. salmonae, or following the use of prototype whole-spore vaccines. Brook trout, in contrast, are unable to mount any protective response upon re-exposure (Speare et al.

2003).

A hypothesis that might explain the episodes of MGDS occurring later in the Chinook

salmon production cycle is that low dose initial exposure L.to salmonae, as might occur when netpen fish are infected from native salmon near netpen sites, may not be sufficient to induce

functional protection to subsequent L. salmonae challenge. Thus, salmon in their second summer of production remain vulnerable to infection. This hypothesis will be tested using a surrogate species (O. mykiss) and a donor-recipient cohabitation infection model to create a group of fish lightly infected withL. salmonae. These fish will then be subsequently challenged with a high dose of L. salmonae to determine their level of protection relative to naive controls.

51 2 3 MATERIALS AND METHODS

2 J.l Sample Population

Juvenile rainbow trout weighing 30-40 grams were purchased from a certified disease- free (notifiable pathogens) commercial hatchery on Prince Edward Island, with no previous history ofL. salmonae. All procedures were conducted in accordance with the guidelines of the

Canadian Council on Animal Care (CCAC 2005).

23 .2 Experimental Design

Eighty-one rainbow trout were randomly selected and housed in a 250 L, flow through, circular fiberglass tank that was supplied from a well water source, maintained at 15.0° C (±0.3°

C). The tank had constant aeration with flow rates maintained at 9 L min'1, with a turnover time of 28 minutes. After a 2 week period of acclimatization, the rainbow trout were fin clipped and challenged by the addition of 12 rainbow trout heavily infected withL. salmonae to serve as donor fish for a period of exposure of 4 weeks. Beginning 4 weeks post exposure recipient fish were anaesthetized using 60 mg/L benzocaine and were screened once per week for the next 4 weeks. Screening consisted of lifting the operculum to permit examination of the entire surface of the first left gill arch for evidence of xenomas using a dissecting microscope. Fish were then placed in water that did not contain benzocaine and allowed to fully recover and were returned to their tank. Screening was done until the gills no longer showed evidence of xenomas, demonstrating a recovery from a primary infection withL. salmonae. These fish were now deemed “low dose recovered.”

Re-challenge Exposure

The 81 low dose recovered fish were divided and randomly assigned in groups of 38 and

43 fish into two 250 L tanks. To each tank 20 naive rainbow trout from the same source were added to serve as positive (defined as not previously exposed L.to salmonae) controls. This trial was carried out over a 15 week period.

52 2.3.3 Methods of Infection

Six weeks prior to the experimental challenge day, a separate population of fish was

infected and once fish displayed numerous mature xenomas they were euthanized by overdose with 100 mg/L benzocaine. The approximate dose of spores generated from this is described in

section 2.3.4. Gills from the euthanized fish were dissected free, finely minced, diluted with tank

water and introduced to the tanks of experimental fish after these fish underwent a 2-day fast.

This introduced material served as infective material as described by Kent et al. (1995). All

material was used immediately following harvest. The water flow to the tanks receiving infected

gill material was turned off for 1 hour to enhance contact of the parasite with the fish (Kent et al.

1995); fish were observed to consume the introduced gill material.

23.4 Dose Quantification

The first left gill arch of fish being used as the source of infective material was non-

lethally examined under a dissecting microscope for branchial xenomas. Upon detection of peak

infection time (defined by xenoma numbers peaking) these fish were euthanized by benzocaine

overdose (100 mg/L) and the first left branchial gill arch was dissected free for whole mount

assessment so that the numbers of xenomas could be counted with the aid of light microscopy.

The total weight of macerated gill material used in this trial was 28.2 grams and this contained

14,816 xenomas. This was the infective material used in this trial, with half given to each of the

two tanks.

235 Sampling and Infection Assessment

During the trial, experimental fish were assessed weekly for the presence or absence of branchial xenomas using the methodology previously described (Speare et al. 1998a). Fish were anaesthetized using 60 mg/L benzocaine. Using a dissecting microscope, all visible gill lamellae were examined. By week 5 post secondary exposure (week 15 in total), xenomas were detected

53 on the majority of the fish, and they were then euthanized using an overdose of benzocaine (100 mg/L), after which the first left gill arch was dissected free, prepared as a whole mount, and the entire structure was examined using a light microscope, and a count of xenomas was made. From these xenoma counts, the mean number of xenoma counts per gill arch (XCPGA) was calculated.

2.3.6 Data Analysis

The data set was assessed in two ways. Chi-square tests of the proportions of fish with xenomas and those without, (Tanks A and B separately, as well as the two tanks combined) were employed to compare the proportion of fish positive for xenomas in both the naive controls and the low dose recovered groups. This was done to test the null hypothesis that infection status

(whether a fish was xenoma positive or negative at various points in the trial), was no different for fish that had recovered from a low dose primary infection compared to control fish that had never been previously infected. A second analysis, using a two-way analysis of variance

(ANOVA) was used to 1) compare the XCPGA results, and 2) test for interations between factors.

Comparison of XCPGA results was employed to see if there were differences in severity of infection as described by xenoma numbers. Specifically this analysis tested the null hypothesis that the numbers of xenomas forming on the gills of fish, subsequent to a high dose Loma exposure, did not differ between those which had recovered from primary low dose infection compared to naive control fish. Interaction was considered and tested adopting a null hypothesis that there would be no subsequent tank interaction.

A one-way ANOVA was used to test the null hypothesis that there would be no difference in mean number of xenomas in both control and low dose recovered groups. Prior to administering the test, the non-zero data was natural log transformed to meet the model’s assumptions of normality (Moore & McCabe 2006).

54 All tests were employed to compare treatments against controls. All statistical analyses were carried out using commercial software (MINITAB™ Inc., Version 15, Pennsylvania, US).

Differences were considered significant at the a<0.05 level of probability.

2.4 RESULTS

The proportion of fish that were completely free of xenomas was significantly higher in groups that had recovered from a primary low dose infection than the control groups in both tanks

(p<0.001), thereby rejecting the null hypothesis. After analysis of the non-zero data set, a total of

95-100% of the fish in the control groups and 47-55% of the fish in the re-exposure groups in both tanks that received oral exposure to L. salmonae-infected gill tissue at 15° C developed xenomas by week 5 and had branchial pathology typical for previously reportedL. salmonae infections (Speare et al. 1998a).

When the XCPGA was compared, it showed that both groups of low dose recovered fish had far fewer xenomas (XCPGA reduced by 89.4% and 93.6%, respectively) as compared to the control groups of fish (Table 2.1).

The two-way ANOVA used to test treatment effect indicated an overall significant difference (p<0.001), evidence that there is a difference between the level of infection in control and low dose recovered groups. Analysis by the two-way ANOVA, employed to deduce any tank interactions, indicated an overall significant difference (p=0.0260); this reflected a higher

XCPGA within fish in Tank A. Although both tanks in the present trial received the same amount (in grams) of infective material, the quantification of number of infective spores present in macerated tissue was not performed as may have led to one tank receiving a slightly higher dose of xenomas. Although this could be indicative of the only difference between the tanks, it does not dismiss concern that tank interaction was indeed the reason for the difference. From the

55 results indicating tank effect, tanks were dealt with separately and analyzed using a one-way

ANOVA.

Analysis by the one-way ANOVA comparing controls to the re-exposed fish in Tank A revealed a significant difference (p<0.001), while one-way ANOVA comparing the two groups of fish in Tank B was also significantly different (p<0.001).

56 Table 2.1: Xenoma outcome at week 5, following an experimental challenge with Loma salmonae, in naive vs. low dose recovered fish

Number % Health Fish °/o Fish Mean Reduced Tank Status n Infected Infected XCPGA X enom as St.D ev. A Naive 20 19 95 44.5 0 42.8 LD Rec 38 21 55 8.1 89.4 6.9 B Naive 20 20 100 34.4 0 61.8 LD Rec 43 20 47 4.8 93.6 4.9

Legend: Fish status denotes exposures prior to experimental challenge. LD Rec indicates those fish that underwent and recovered from a primary low dose cohabitation exposure, n = number of fish per group, Mean XCPGA = ((Total xenomas/16)/n), % reduced xenomas = ((XCPGAcontrol - XCPGAtreatment)/(XCPGAcontrol))* 100%, St. Dev. = standard deviation.

57 2.5 DISCUSSION

This is the first study showing that fish which recover from a primary low dose cohabitation exposure toL. salmonae are capable of mounting an overall significant protective response against re-exposure to the parasite ofL. salmonae by means of a high dose oral re­ challenge as the number of xenomas in rainbow trout was significantly reduced. In previous work, Speare et al. (1998b) found that rainbow trout that underwent a primary high dose oral infection and allowed to fully recover were capable of mounting a protective response to a subsequent re-challenge to a secondary high-dose challenge. FurtherL. salmonae resistance studies were completed by Kent et al. (1999) and demonstrated the protective responses upon re­ exposure to the parasite was not only limited to a single species, as Chinook salmon were capable of being fully protected against xenoma formation, while Speare et al. (2003) discovered the inability of brook trout to reduce xenoma burden dining re-exposure.

Compared to a single high dose exposure, a low dose cohabitation exposure more closely mimics the natural route of infection occurring in netpens, which is the shedding of infective spores over a period of time (Shaw et al. 1998; Ramsay, Speare, Becker & Daley 2003). Naive fish in the study would have contacted spores in the environment released from infected cohorts upon the rupturing of mature xenomas. Although there are some difficulties in determining the dose of spores received by naive fish, the probability of successful contact occurring is dependent upon a number of factors such as the concentration of spores in the tank, the population density of susceptible hosts, tank turnover time and water temperature (Reno 1998; Becker, Speare &

Dohoo 2003). With this in mind, tank parameters were set as per previousin vivo experimental studies that have been shown to optimize not only the dissemination of disease and exposure of naive fish to spores, but also to promote completion of the parasite’s life cycle in newly infected individuals (Beaman, Speare & Brimacombe 1999a; Speare et al. 1998c; Speare et al. 1999b ).

58 Naive rainbow trout used in the study were housed in a quarantined fresh water flow through tank maintained at 11°C and were transferred to the challenge tank held at 15°C 2 weeks prior to being subjected to a low dose exposure model. The increased temperature profoundly affects fish metabolism, indirectly increasing oxygen demand in a situation where oxygen solubility has decreased due to temperature changes (Atkins & Benfey 2008). Therefore a lengthy period of acclimation is required as it allows fish to slowly adjust to changes in tank parameters. Since fish undergoing stress tend to have compromised immune systems (Branson

1993; Collins 1993; Southgate 1993; Stoskopf 1993), the period of acclimation employed eliminated rapid temperature change as a stressor during the low dose challenge, thereby optimizing the exposure model.

Prior to cohabitating with naive individuals, the 12 donor fish were examined and categorized as being heavily infected with L. salmonae by non-lethal inspection of the gills using a dissecting microscope. The number of donor fish used was based on their infection status and previous studies on the transmission potential ofL. salmonae that had a ratio of 9:1 naive fish to donor fish, with over 94.5% disease prevalence in cohabitated naive fish (Becker, Speare &

Dohoo 2005). In comparison, our challenge model went above and beyond that with approximately a 7:1 ratio. Furthermore, the xenoma intensity noted within the donor rainbow trout was indicative that this population of rainbow trout acquired from a local hatchery was never exposed toL. salmonae, as their infections were characteristic of being originally naive to the parasite (Speare et al. 1998a; Ramsay et al. 2003). After addition to the tank, screening of the donor fish was performed on a weekly basis to ensure disease dissemination (i.e. the xenomas were indeed rupturing) by examining the first left gill arch for a period of 4 weeks post cohabitation. Once the gills of the donor rainbow trout were free of intact xenomas thereby indicating the end of infection, they were humanely euthanized by means of benzocaine overdose

(lOOmg/L).

59 During the oral challenge exposure with 14.1 grams of infected material per tank, flows were turned off to both tanks to promote contact of fish with the parasite. These parameters were derived from a study by Kent et al. (1995), employing a similar exposure model with 5 grams of infected material, at which time flows were halted for a period of 2 hours. The decision to decrease exposure time in this study was based upon the amount of material used for the challenge, which more than doubles that delivered in the transmission study by Kent et al. (1995).

From the above it has been concluded that the low dose co-habitation challenge method and high dose challenge method were comparable to methods used in previous studies.

Many studies, including those initially performed forL. salmonae employed an artificial infection model by either introducing the parasite orally (Speare et al. 1998a), intraperitoneally

(Shaw et al. 1998) or by other means. Although these methods can easily manage the number of xenomas being delivered and the exposure duration, they are less natural than cohabitation with infected cohorts. Delivering an oral exposure to naive fish gives researchers the ability to quantify the number of xenomas being delivered to experimental tanks, but does not allow for the quantification of xenomas being delivered to each individual fish as feeding is variable and is closely associated with water temperature, tank hierarchy, aggression, competition between fish, hunger, individual stomach capacity and the appetence of the viscera (Brett 1979; Southgate

1993). Although the cohabitation model does not allow for an estimate of the number of infective spores being ingested, the dispersal of the parasitic spores throughout the population is not dependent upon behavioral and feeding responses that have long been recognized in fish. In fact, it is somewhat of an unknown how naive fish make contact with spores that are likely floating freely in the water column.

Comparisons between infection models are frequently the object of scientific study. For example, in a study investigating the role of exposure models of the hepatitis E virus affecting swine, a natural model of exposure (contact with infected pigs) was compared to a less natural exposure (intravenous injections). The results revealed differences in the course of infection, in

60 that pigs undergoing cohabitation exposure had longer incubation periods and shorter disease duration than those that had received the virus intravenously, leading researchers to believe that the cohabitation model most likely mimics what is occurring in a natural setting (Bouwknegt,

Rutjes, Reusken, Stockhofe-Zurwieden, Frankena, de Jong, de Roda Husman & Poel 2009). The differing lengths of incubation time reflected in the previous study is congruent with that ofL. salmonae, in the study by Ramsay et al. (2003) in which it was demonstrated that an oral exposure results in an acute infection with typical clearance of disease (rupturing of xenomas) occurring in 10 weeks, while the infection induced by means of cohabitation with infected tank mates was considered slightly more chronic by lasting 12 weeks post exposure.

Although it is Chinook salmon reaching the marketable stages between 2.9 and 4.0 kilograms that are most profoundly impacted by severe mortalities due to infection withL. salmonae in marine netpens, the present model employed rainbow trout between 30-40 grams.

(M. Tchipeff, Creative Salmon Inc. 2008, personal communication). As demonstrated in various studies, infection with the microsporidianL. salmonae runs a similar course regardless of the size of the individual, as both the use of rainbow trout ranging in size from 4-174 grams (Speare et al.

1998a) and Chinook salmon ranging 12.9-119.2 grams (Shaw et al. 1998 & Kent et al. 1995) had the ability to become infected with the parasite by similar means of exposure, and xenomas formed on the gills at the same rate.

Peak infection time in the trial was assumed to be 5 weeks post exposure to the oral challenge as per previous studies indicating that formation of xenomas occurs during this time with dissemination happening between 7-10 weeks post exposure to infected material (Speare et al. 1998a). However, some fish examined lacked xenomas within the gill filaments and therefore the week chosen may have led to an inaccurate estimation of the true potential of MGDS. In future studies, the time at which the fish are culled should be taken into further consideration, perhaps by determining the level of spore maturity by means of electron microscopy (Rodriguez-

61 Tovar et al. 2002; Rodriguez-Tovar et al. 2003), in order to establish a period of time at which the

infection is at peak intensity immediately prior to xenoma dissolution.

The method of initial exposure affected the protective response rainbow trout were able to raise against re-exposure via a high dose oral challenge. Fish that received an initial high dose

challenge were capable of full protection against the onset of MGDS, while those that underwent

a low dose cohabitation exposure demonstrated partial resistance. Similarly, the experimental

cohabitation of naive Atlantic salmon with tank mates infected with amoebic gill disease (AGD)

showed that previously exposed fish were able to mount protective responses upon re-exposure of

the same method to AGD, with 68% of the re-exposed fish having a decrease in active lesions as

compared to control fish exhibiting 100% infection with increased abundance of lesions

associated with AGD (Findlay, Helders, Munday & Gurney 1995). Much like the current

findings concerning L. salmonae, partial protection arose from an initial low dose cohabitation

infection upon re-exposure.

The current study, demonstrated that rainbow trout which recover from a light infection

of L. salmonae, acquired through a cohabitation exposure process are well protected from a

subsequent high dose exposure; given the previously detailed similarities in responses of rainbow trout and Chinook salmon, it is reasonable to anticipate that these results would extend to

Chinook salmon. If so, the question of why Chinook salmon in netpen aquaculture scenarios develop severe MGDS late in the course of their production cycle remains a mystery. The expectation, based on the current study, is that farmed Chinook salmon, which likely would be acquiring low dose infections from horizontal transmission from wild salmon, would acquire a sufficient level of immunity such that MGDS outbreaks late in production would not be occurring. However, this seems not to be the case; several theories can be developed, and these could form the basis for future work in this area. For example, although Chinook salmon acquire protective immunity following a high dose exposure, it is possible that this species differs from rainbow trout in not developing protective immunity from low dose exposures. To date, this has

62 not been directly studied. Alternatively, immunity may develop, but it is of insufficient duration to protect salmon at a later point in time when they may be exposed to a high dose. The current study has not looked at the duration of acquired immunity from a low dose challenge, although other studies with rainbow trout have shown that immunity developing after either vaccination, or high dose challenge can persist for many months (Speare et al. 1998b; Beaman et al. 1999b;

Sanchez et al. 2001c; Rodriguez-Tovar et al. 2006a; Rodriguez-Tovar et al. 2006b; Speare et al.

2007). Finally, the possibility exists that the assumption that farmed Chinook salmon are exposed to L. salmonae from wild carriers, at an early part of the netpen production, may be incorrect.

The release of spores from wild salmon withL. salmonae may either be at too low a level, or become sufficiently diluted, that nearby fanned salmon do not become infected by this.

Therefore, they would remain unexposed and not acquire protective immunity. However, later in production they do become infected; the source of theL. salmonae has been assumed as derived from wild reservoirs. The question then might be: why is exposure from wild reservoirs functionally limited to the time period when farmed salmon are approaching market size? The basis for this may lie in the size of the mesh that is used for netpen salmon cages. When the salmon are young, and newly introduced to the ocean, the net mesh size is quite small. This could exclude certain age classes of wild salmon from entering the pens. Later in production, when cage mesh size is increased, this allows wild salmon to enter productions pens. Perhaps this closer cohabitation, combined with the potential for fanned salmon to eat small wild salmon that have entered the netpens, may result in the relatively high dose ofL. salmonae being encountered by farmed salmon at this latter part of their production cycle.

63 2.6 References

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BEAMAN HJ, SPEARE DJ & BRIMACOMBE M. Regulatory effects of water temperature on Loma salmonae (Microspora) development in rainbow trout. Journal of Aquatic Animal Health 1999a; 11:237-45.

BEAMAN HJ, SPEARE DJ, BRIMACOMBE M & DALEY J. Evaluating protection against Loma salmonae generated from primary exposure of rainbow trout,Oncorhynchus mykiss (Walbaum), outside of the xenoma-expression temperature boundaries. Journal of Fish Diseases 1999b; 22: 445-50.

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BECKER JA, SPEARE DJ & DOHOO IR. Effect of water temperature and flow rate on the transmission of microsporidial gill disease caused byLoma salmonae in rainbow trout Oncorhynchus mykiss. The Japenese Society of Fish Pathology 2003; 38:105-112.

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64 JARP J, GJEVRE AG, OLSEN AB & BRUHEIM T. Risk factors associated with furunculosis, infectious pancreatic necrosis and mortality in post-smolt of Atlantic salmon,Salmo salar L. Journal of Fish Diseases 1995; 18: 67-78.

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KENT ML & POPPE TT. Fungi and Related Organisms. In: Kent ML & Poppe TT, eds. Diseases of Seawater Netpen-Reared Salmonid Fishes. British Columbia: Pacific Biological Station, 1998b: 46-8.

KENT ML, TRAXLER GS, KIESER D, RICHARD J, DAWE SC, SHAW RW, PROSPERI- PORTA G, KETCHESON J & EVELYN TPT. Survey of Salmonid Pathogens in Ocean-Caught Fishes in British Columbia, Canada. Journal of Aquatic Animal Health 1998a; 10: 211-19.

LINDENSTR0M T, COLLINS CM, BRESCIANI J, CUNNINGHAM CO & BUCHMANN K. Characterization of a Gyrodactylus salaris variant: infection biology, morphology and molecular genetics. Parasitology 2003; 127: 165-77.

LOM J. A catalog of described genera and species of microsporidians parasitic in fish. Systematic Parasitology 2002; 53: 81-99.

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RAMSAY JM, SPEARE DJ, BECKER JA, DALEY J. Loma salmonae-associated xenoma onset and clearance in rainbow trout, Oncorhynchus mykiss (Walbaum): comparisons ofper os and cohabitation exposure using survival analysis. Aquaculture research 2003; 34: 1329-35.

RENO PW. Factors involved in the dissemination of disease in fish populations. Journal of Aquatic Animal Health 1998; 10: 160-71.

RODRIGUEZ-TOVAR LE, BECKER JA, MARKHAM RJF & SPEARE DJ. Induction time for resistance to microsporidial gill disease caused byLoma salmonae vaccination of rainbow trout (iOncorhynchus mykiss) with a spore-based vaccine. Fish & Shellfish Immunology 2006a; 21: 170-75.

65 RODRIGUEZ-TOVAR LE, MARKHAM RJF, SPEARE DJ & SHEPPARD J. Cellular immunity in salmonids infected with the microsporidial parasiteLoma salmonae or exposed to non-viable spores. Veterinary Immunology and Immunopathology 2006b; 114: 72-83.

RODRIGUEZ-TOVAR LE, WRIGHT GM, WADOWSKA DW, SPEARE DJ & MARKHAM RJF. Ultra-structural study of the early development and localization Lomaof salmonae in the gills of experimentally infected rainbow trout. Journal of Parasitology 2002; 88: 244-53.

RODRIGUEZ-TOVAR LE, WRIGHT GM, WADOWSKA DW, SPEARE DJ & MARKHAM RJF. Ultra-structural study of the late stages ofLoma salmonae development in the gills of experimentally infected rainbow trout. Journal of Parasitology 2003; 89: 464-74.

SANCHEZ JG, SPEARE DJ, MARKHAM RJF & JONES SRM. Experimental vaccination of rainbow trout against Loma salmonae using a live low-virulence variant of Loma salmonae. Journal of Fish Biology 2001c; 59:442-48.

SHAW RW, KENT ML & ADAMSON ML. Modes of transmission of Loma salmonae (Microsporidia). Diseases of Aquatic Organisms 1998; 33: 151-56.

SOUTHGATE P. Disease in aquaculture. In: Brown L, ed. Aquaculture for Veterinarians: Fish Husbandry and Medicine. Oxford: Pergamon Press, 1993: 91-129.

SPEARE DJ, ARSENAULT GJ & BUOTE MA. Evaluation of rainbow trout as a model for use in studies on pathogenesis of the branchial microsporidianLoma salmonae. Contemporary Topics 1998a; 37: 55-8.

SPEARE DJ, BEAMAN HJ & DALEY J. Effect of water temperature manipulation on a thermal unit predictive model forLoma salmonae. Journal of Fish Diseases 1999b; 22: 1-7.

SPEARE DJ, BEAMAN HJ, JONES SRM, MARKHAM RJF & ARSENAULT GJ. Induced resistance in rainbow trout, Oncorhynchus mykiss (Walbaum), to gill disease associated with the microsporidian gill parasiteLoma salmonae. Journal of Fish Diseases 1998b; 21: 93-100.

SPEARE DJ, BRACKETT J & FERGUSON HW. Sequential pathology of the gills of coho salmon with a combined diatom and microsporidian gill infection. Canadian Veterinary Journal 1989; 30:571-75.

SPEARE DJ & DALEY J. Failure of vaccination in brook trout Salvelinus fontinalis against Loma salmonae (Microspora). Fish Pathology 2003; 38: 27-8.

SPEARE DJ, DALEY J, MARKHAM RJF, SHEPPARD J, BEAMAN HJ & SANCHEZ JG. Loma salmonae-associated growth rate suppression in rainbow trout,Oncorhynchus mykiss (Walbaum), occurs during early onset xenoma distribution as determined byin situ hybridization and immunohistochemistiy. Journal of Fish Diseases 1998c; 21: 345-54.

SPEARE DJ, MARKHAM RJF & GUSELLE NJ. Development of an effective whole-spore vaccine to protect against microsporidial gill disease in rainbow trout (Oncorhynchus mykiss) by

66 using a low-virulence strain of Loma salmonae. Clinical and Vaccine Immunology 2007; 14: 1652-54.

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XU DH, KLESIUS PH, PANANGALA VS. Induced cross-protection in channel catfish, Ictalurus punctatus (Rafinesque), against different immobilization serotypes ofIchthyopthirius multifiliis. Journal of Fish Diseases 2006; 29: 131-38.

67 3. FIRST DEMONSTRATION OF THE PARTIAL EFFICACY OF A HETEROLOGOUS VACCINE AGAINST LOMA SALMONAE , USING SPORES FROM TWO SPECIES OF GLUGEA.

3.1 ABSTRACT

As an extension of prior work in which it was demonstrated that experimental spore-based vaccines from virulent and avirulent strains of L. salmonae could induce protective responses in rainbow trout against experimental challenges withL. salmonae, the current study examines the feasibility of developing heterologous vaccines based on whole live spores from two species of microsporidia from a genus closely related toLoma (Glugea hertwigi and Glugea anomala). To

examine the efficacy of this alternative vaccination approach, five groups of fish were used including: a naive unvaccinated group (positive control), a group which had recovered from a prior high dose initial challenge with L. salmonae (high dose recovered), a group which received a low dose initial challenge with L. salmonae (low dose recovered), a group which received a single injection with live G. hertwigi spores (G. hertwigi vaccinated), and a group which received a single injection with live G. anomala spores (G. anomala vaccinated). Upon recovery from

initial challenge (Loma groups), and six weeks following vaccination (Glugea vaccinated groups), all fish were orally re-challenged with a virulent strain ofZ,.salmonae ; five weeks later the fish were euthanized and the infection status (based on presence or absence of xenomas), and mean numbers of xenomas per gill arch were determined and compared to the unvaccinated control group. Whereas 87% of the naive unvaccinated control fish developed xenomas, the proportion of fish with xenomas was reduced by 100% in the high dose recovered group, 58% in the low dose recovered group, 29% in the G. hertwigi vaccinated group, and 24% in the G. anomala vaccinated group. Compared to the control group, all of these differences were statistically significant. More dramatically, when the xenoma burden was compared against the naive controls, the reduction in xenoma burden was 100% in the high dose recovered group, 12% in the low dose recovered group, 81% in the G. hertwigi vaccinated group, and 82% in the G.

68 anomala vaccinated group. Compared to the control group, all of these differences were statistically significant. Based on the striking reduction in xenoma burden in the vaccinated groups, this demonstration of the efficacy of a heterologous vaccine againstL. salmonae justifies further work in this area. \

3.2 INTRODUCTION

As a significant disease of marine netpen cultured Chinook salmon (O. tshawystcha) along coastal British Columbia, Microsporidial Gill Disease of Salmon (MGDS), caused by the pathogen L. salmonae (Hauck 1984; Kent et al. 1989; Speare et al. 1998a; Sanchez et al. 2001b) presents several challenges with respect to developing practical health management strategies.

Striking in late summer or early fall, outbreaks most often affect fish that are nearing market size

(Kent et al. 1989), and thus are more valuable. Additionally, although several drug treatment options are showing promise (Kent et al. 1994; Speare et al. 1998d; Speare et al. 1999a; Speare et al. 2000; Sanchez 2001c; Becker et al. 2002; Becker & Speare 2004; Guselle et al. 2006; Guselle et al. 2007; Rodriguez-Tovar 2006a,b; Speare et al. 2007) application of these treatments, even once they have become licensed for this purpose, will be hindered because withdrawal times could affect timing of fish harvest. The question of why the disease strikes fish in their second summer of production remains unanswered and the trend seems paradoxical particularly since it has been repeatedly shown that in experimental models of MGDS, involving several species of salmonid including the principle species of concern, that recovery from infection leads to protective immunity (Speare 1998b; Kent et al. 1999). Taken together, these findings would suggest that near-market salmon, having been at sea for many months and likely exposed toL. salmonae from natural reservoirs (Kent et al. 1989), would have developed protective immunity before they reached their final grow out stage. However, the opposite is described by aquaculturists (T. Rundle, Creative Salmon Inc. 2008, personal communication). It was hypothesized that whereas experimental studies have used a high dose initial challenge, the discrepancy between laboratory findings and on-farm observations might suggest that a low-dose initial challenge, as could happen when farmed fish become lightly infected through cohabitation with infected cohorts or from picking up light infections from nearby wild fish that are carriers, provides an insufficient antigen challenge to provoke protective immunity. However, results in

Chapter 2 do not support this hypothesis and it appears, at least in the rainbow trout model of this disease, that low dose exposure acquired through co-habitation is sufficient to induce near complete protection even against a high dose secondary challenge. Although this was unexpected, the findings nevertheless underscore the potential value of further developing vaccination strategies for this disease.

The use of vaccines is well established in aquaculture and commercial products ranging from bacterins to subunit and DNA-based preparations are available against many significant bacterial and viral diseases of salmonids and non-salmonids. However, to date, there are no commercial vaccines against parasitic or fungal diseases of fish despite the importance of these two large groups of pathogens in aquaculture settings. Microsporidians have characteristics that might place them into either the category of fungus or parasite, with the debate stemming from the 19* century discovery ofNosema sp. and the consideration of this microsporidian to be a

“yeast-like fungus”, at which time the concept of protists were in their infancy (Keeling at al.

2002). Cavalier-Smith (1993) proposed that microsporidians were actually primitive eukaryotes; based on their lack of mitochondria, flagella and other 9+2 structures, they were designated as belonging to the Kingdom Archezoa, most commonly defined as amitochondrial organisms

(Cavalier-Smith 1993; Keeling et al. 2002). With the advancing methods of molecular phylogenetics, support was given to the Archezoa hypothesis as microsporidia demonstrated retention of the prokaryotic trait of having their 5.8S rRNA fused to the large subunit (LSU) rRNA (Vossbrinck et al. 1987). Despite the accumulated evidence, the categorization of microsporidia was still questionable due to their highly specialized parasitic lifestyle and the

70 ability of their minute rRNA to undergo several unique deletions, suggesting that the LSU-fused

5.8S rRNA found in microsporidia may be the result of improper enzyme recognition and

cleavage due to evolutionary reduction, and not as the result of retention of prokaryotic .traits

(Cavalier-Smith 1993). It was with the discovery of sequences in species of microsporidia

corresponding to both alpha- and beta-tubulins that the relatedness of these amitochondrial

species to fungi was demonstrated (Keeling & Doolittle 1996; Edlind, Li, Visvesvara, Vodkimn,

McLaughin & Katiyar 1996), marking one of many contradictions to the Archezoa hypothesis.

Although phylogenetic analyses are limited due to inadequate amounts of fungal representatives,

researchers have been able to tie microsporidia to fungi through their respective meiotic

mechanisms, mRNA capping mechanisms and use of a closed spindle formation (Gill & Fast

2006). Microsporidia have been shown to express a heat shock protein 70 (HSP70) gene,

demonstrating the retention of a mitochondrial characteristic, further demonstrating relatedness to

the fungal world (Keeling et al. 1996).

There has been limited success in developing effective vaccines against parasites or

fungi. An important challenge is overcoming antigenic variation. For example, subunit vaccines

for malaria, which utilize the dominantly expressed antigen, confer only a limited level of protection; development of a whole cell vaccine may be more effective (Vaugh, Wang & Kappe

2010). In support of this, effective prototype whole spore vaccines againstL. salmonae have been developed (Speare et al. 2007).

To date, there has been very limited success with developing vaccines for diseases caused by microsporidians. Of the microsporidians infecting mammals, progress towards understanding the protective response againstEncephalitozoon spp. has provided encouraging results. Studies

involving the passive transfer of hyperimmune antiserum failed to protect mice from lethal disease, but may be involved in the opsonization of microsporidia by macrophages and induction of complement-mediated killing of the parasite (Schmidt & Shadduck 1984). Since the

71 development of antibodies against microsporidia is variable, no information has been published that describes the functions of microsporidia-specific antibodies in humans (Didier, Snowden &

Shadduck 1998). Studies involving athymic mice have demonstrated that the type of response is cell-mediated as those that undergo adoptive transfer of sensitized T lymphocytes are protected while those that do not end up succumbing to the disease (Didier et al. 1998; Weiss et al. 1999).

Further support for a T-cell immune response toE. cuniculi is seen in immunocompromised patients with T-cell deficiencies who suffer from more severe clinical signs than those considered

immunologically competent (Didier et al. 1998; Asmuth et al. 1994). Studies on the

immunoprotective responses of trout toL. salmonae add further support to the idea that cell- mediated responses are key (Rodriguez-Tovar et al. 2006b). Based on previous work, die response is thought to be cell-mediated in that xenoma rupture causes proliferation of mostly head kidney mononuclear cells (MNC) to antigens being released. Although cell mediated studies on microsporidians is still in its infancy, Rodriguez-Tovar et al. (2006b) demonstrated that throughout all the stages of infection with L. salmonae, MNC proliferation was observed to be

Lorna-specific as unexposed animals had no proliferative response. Initial responses to oral or intraperitoneal (IP) injection exposure were able to induce a proliferation of MNC, while a moderate proliferation was noted in orally re-exposed animals that had initially received an oral challenge and a significant proliferation was seen in those that had received an initial IP challenge of dead spores. The varying levels of responsiveness to live and dead spores is questionable but is thought to be influenced by the chitinous cell wall. Chitin and its derivatives have been demonstrated to induce cytokine release from T cells, increase phagocytic activity and enhance immune responses to various diseases in fish and mammals (Suziki, Okawa, Hashimoto, Suzuki

& Suzuki 1984; Nishimura, Nishimura, Nishi, Saiki, Tokura & Azuma 1984; Nishimura,

Nishimura, Nishi, Numata, Tone, Tokura & Azuma 1985; Sakai, Kamiya, Ishii, Atsuta &

Kobayashi 1992) and therefore a quick cell mediated response. Since the delivery of dead spores intraperitoneally would create a large bolus of chitinous material, it is easy to imagine a

72 significant proliferative response, while the oral delivery of spores to animals may not have the same dramatic effect, due to the limited knowledge of the exact nature of the pathogen as it transits through the gut to the heart and into the gills (Rodriguez-Tovar 2006a,b). From this, several experimental prototype vaccines have been evaluated under laboratory conditions. Initial studies used both a dose of fresh spores or a dose of freeze-killed spores and found that although able to confer complete resistance against an experimental oral challenge, injections with fresh spores ofL. salmonae caused formation of xenomas within gill vasculature, while fish that received an injection of freeze-killed spores did not elicit xenoma formation and were incompletely but significantly protected when experimentally challenged compared to controls

(Speare et al. 1998b). Even though the live vaccine provides a robust immunity, it does present safety risks, such as allowing a full scale infection to occur within the vaccinated population and potential spread to unvaccinated cohorts. It is with this in mind that further studies investigated the ability of intraperitoneal delivery of spores harvested from brook trout heavily infected with a low virulence strain of L. salmonae (SV strain) that underwent freeze-kill were able to significantly limit MGDS in rainbow trout against an experimental oral challenge with the virulent OA strain (Rodriguez-Tovar et al. 2006a; Speare et al. 2007).

Work to date with prototype vaccines has been based on the use of whole-spore preparations as they are generally recognized as being more protective, perhaps due to optimal presentation of antigens, thereby having the ability to elicit immune responses against multiple antigenic targets (Skoble, Beaber, Gao, Lovchik, Sower, Liu, Luckett, Peterson, Calendar,

Portnoy, Lyons & Dubensky 2009). Since few advances have been made in the case of effective eradication strategies against microsporidial diseases, the idea of using whole spores for experimental vaccines is quite an understandable and valid approach, as the chances of antigenic recognition are increased. Experimental approaches for vaccines against the causative agent of

Anthrax, Bacillus anthracis, has through the years employed antigen specific vaccines, such as the human vaccine containing the single protective antigen (PA), with encouraging results, but researchers are discouraged by undefined composition, lot-to-lot variation and the extensive

dosing regimes that are required with these formulations (Friedlander & Little 2009). It has been

shown that killed but metabolically active (KBMA) whole spore vaccines in animal models is

generally more protective against anthrax and is believed to be the case due to the inclusion of a broad spectrum of antigens. Work that has been completed on KBMA whole spore vaccines have

shown the promise of their ability to allow for fewer doses, a more rapid and enhanced immune response due to broader repertoires of immune cells being elicited and a greater ease of use overall, which increases their marketability (Friedlander et al. 2009; Skoble et al. 2009).

Given the results obtained to date with a whole spore vaccine, particularly the killed vaccine developed from the avirulent strain, it might be possible to begin work towards commercialization. However, several challenges remain and a key roadblock becomes the production of spores in quantities that could be used by commercial vaccine manufacturers.

Although the virulent and avirulent strains of Loma can be produced in reasonable quantity through planned infections of various species of trout, and harvested from the gills when xenomas have become abundant, this is unlikely to be a technological strategy that would be adopted by a commercial vaccine company. An alternative, growingLoma in cell culture and harvesting various life stages, would hold promise, but there has been limited success in growing microsporidians on cell lines. Much success has been shown in culturing microsporidial species ft that infect insects and humans, with some isolates being grown and maintained easier than others

(Monaghan, Kent, Watral, Kaufman, Lee & Bols 2009). Microsporidia known to infect humans can be successfully infected as finite or continuous cell lines and are maintained for months or years (Monaghan et al. 2009). On the other hand, cell culture of fish-affecting microsporidia can only be maintained as primary cultures, lasting about 48 hours. These short-term cultures are primarily used to investigate macrophage and neutrophil interactions with the parasitic spores.

74 Much of the success seen in the cultivation of the stages of microsporidia is noted in long term primary cultures and not so in cell culture (Monaghan et al. 2009). The evidence suggests that it is not the lack of fish cell lines, but it is perhaps that microsporidia require specific differentiated cell types from fish in order to grow successfully in culture. With this in mind, it is hypothesized that an alternative approach could involve investigating the use of a heterologous vaccine where the source of antigen could allow us to overcome some production limitations.

The close phylogenetic relationship ofGlugea to Loma would suggest that spores from species in this genus conceivably could generate cross protection. BothLoma and Glugea are morphologically similar with each being polysporoblastic, with sporogony occurring inside a septated parasitophorous vacuole as well as being monokaryotic through all developmental stages

(Nilsen 2000). Additionally, in a phylogenetic study conducted by Brown, Kent and Adamson

(2010), species within Glugea form well supported clusters with bootstrap values exceeding 99%, while those within Loma are paraphyletic, but form monophyletic clusters within their own genus with bootstrap values of 100%. This would suggest that although having overlapping physical characteristics, that Glugea and Loma are two distinctly different species of microsporidians affecting aquaculture and may have the capability of generating cross protection. There might be several advantages to using Glugea. If we consider harvesting of spores from infected animals,

Glugea typically produces exceptionally large xenomas. The cysts are macroscopic and laden with mature spores that are easily harvested by means of careful dissection of the apparent whitish xenoma from affected body tissue or viscera. Spores can be released from xenomas by means of mechanical disruption in order to be purified. Furthermore, Glugea spp. are one of the few fish-affecting microsporidians to be maintained in continuous cell culture on larval cells of the mosquito (Aedes albopictus). With the ability to produce new virulent spores in excellent yields in a 72-hour window, this culture allows for a potential short-term production scenario

(Lores, Rosales, Mascaro & Osuna 2003).

75 The affinity to infect the gastrointestinal tract (Scarborough & Weidner 1979), clinical signs upon dissolution of spore filled xenomas and the gravest number of mortalities occurring in late summer and early fall are not exclusive toL. salmonae. Seasonal mortality is also observed in Glugea spp. infections (Scarborough et al. 1979), including G. hertwigi (Nepszey, Budd &

Dechtiar 1978). With their ability to infect a wide variety of salmonids, in both farmed and wild, as well as in the laboratory (Lee, Yokoyama & Ogawa 2004), the members of the genusGlugea are therefore readily available to study the potential of cross-protection against infection withL. salmonae.

To date, no studies have examined the potential for heterologous vaccines to protect against microsporidian infections. The objective is to determine whether a vaccine against L. salmonae could be developed using spores harvested from xenomas of twoGlugea species. This was tested using a surrogate species (O. mykiss), undergoing a high dose challenge after vaccination in order to determine their level of protection relative to naive controls. Furthermore, a re-examination of the level of protective response generated in Chapter 2 from a recovery from a low dose cohabitation exposure upon receiving a high dose oral re-exposure.

3 3 MATERIALS AND METHODS

3 J.l Sample Population

Juvenile rainbow trout weighing 30-40 grams were purchased from a certified disease- free (specific pathogens) commercial hatchery on Prince Edward Island, with no previous history ofL. salmonae. All procedures were conducted in accordance with the guidelines of the

Canadian Council on Animal Care (CCAC 2005).

76 3.3.2 Experimental Design

Low Dose Primary Exposure

A group of heavily infected fish were housed separately during acclimatization of 58 naive rainbow trout in a separate 250L, flow through, circular fiberglass tank that was supplied from a well water source, maintained at 15.0° C (±0.3° C). The 2-week acclimating period allowed for non-lethal inspection of donor fish gills using a dissecting microscope, of which 6 were deemed heavily infected withL. salmonae. The number of donor fish used for cohabitation was based on their infection status and previous studies on the transmission potential ofL. salmonae that had a ratio of 9:1 naive fish to donor fish. This exact ratio was employed and after addition to the tank, screening of the donor fish was performed on a weekly basis to ensure disease dissemination by examining the first left gill arch for a period of four weeks post cohabitation. Once the gills of the donor rainbow trout were free of intact xenomas, fish were indicated as coming to the end of infection and disease dissemination. The donor fish were then humanely euthanized by means of benzocaine overdose (lOOmg/L).

Beginning 4 weeks post exposure recipient fish were anaesthetized using 60mg/L benzocaine and were screened once per week for 4 weeks. Screening consisted of lifting the operculum and the examination of the entire surface of the first left gill arch for evidence of xenomas using a dissecting microscope. Fish were then placed in water that did not contain benzocaine and allowed to fully recover and were returned to their tank. At this time 19 of the 58 fish were considered positive for infection by visually detecting xenomas on the first left gill arch.

These infected fish were then screened until the gills no longer showed physical signs of xenomas, demonstrating a recovery from a primary infection withL. salmonae. These fish were now deemed “low dose recovered.”

77 High Dose Primary Exposure

Twenty naive, juvenile rainbow trout were randomly selected and housed in a 70 L tank with the same water parameters as previously described for the “Low Dose Primary Exjtosure.”

After a 2 week period of acclimatization, naive individuals received macerated gill material heavily infected withL. salmonae from fish that were categorized as highly infected via microscopic examinations that were humanely euthanized by means of benzocaine overdose

(lOOmg/L). The finely minced material was diluted with tank water and introduced to the tank after a 2-day fast. All fish were feeding at the time of exposure and water flow was turned off for

1 hour to ensure contact of the parasite with the fish. Screening was done until the gills no longer showed physical signs of xenomas, demonstrating a recovery from a primary infection L.with salmonae. These fish were now deemed “High Dose Recovered.” During the onset of infection, one fish died for other reasons.

Glugea Vaccine Preparation and Delivery

Sixty rainbow trout were selected and randomly assigned in equal groups to two 70 L tanks and allowed 2 weeks to acclimatize prior to vaccination. Xenomas of G. hertwigi were harvested from wild caught smelts, Osmerus mordex, inhabiting an estuary located in Eastern

Prince Edward Island.

Spores were isolated and purified (Appendix B) and were administered by intraperitoneal injection (IP) to a single tank of 30 fish at a dose of 106 in sterile saline. A second tank of 30 fish were vaccinated via IP injection at a dose rate of 106 with sterile saline and G. anomala spores that were isolated and purified (Appendix B) from xenomas harvested from three-spined sticklebacks, Gasterosteus aculeatus and nine-spined sticklebacks, Pungitius pungitius that were housed at the Atlantic Veterinary College, Prince Edward Island. Both species of sticklebacks were collected by netting from estuaries and near-shore parts of the ocean surrounding Prince

78 Edward Island. The rainbow trout were maintained 6 weeks post exposure and monitored for any

change in health. Fish returned to normal feeding behaviour 48 hows post vaccination and no

mortalities were noted at this time.

Re-challenge Exposure

Prior to re-challenge, the 99 fish (19 “Low Dose Recovered”, 20 “High Dose Recovered”

and 60 “Glugea spp. Vaccinated”) were assigned to a 250 L tank at which time 30 naive rainbow trout from the same fish sowce were added to serve as positive controls. The entire trial was

carried out over a period of 15 weeks.

3.3.3 Methods of Infection

Six weeks prior to the experimental challenge day, a separate population of fish were

infected with macerated gill material infected with L. salmonae and once they displayed

numerous mature xenomas they were euthanized by overdose with 100 mg/L benzocaine. Gills

from the euthanized fish were dissected free, finely minced, diluted with tank water and

introduced to the experimental tank after a 2-day fast to serve as infective material as described by Kent et al.~(1995). All material was used immediately following harvest. The water flow to the tanks receiving infected gill material was turned off for 1 how to enhance contact of the parasite with the fish (Kent et al. 1995).

3 J.4 Challenge Dose Quantification

The first left gill arch for all fish was non-lethally examined under a dissecting scope for branchial xenomas. Upon assumption of peak infection time all fish were euthanized by benzocaine overdose (100 mg/L) and the first left branchial gill arch was dissected free for whole mount and xenomas were counted using a stereomicroscope resulting in a xenoma count per gill arch (XCPGA). The estimated total amount of xenomas delivered to the experimental tank of fish was approximately 4,544 or roughly 35 xenomas per fish.

79 3.3.5 Sampling and Infection Assessment

During the trial, fish were assessed weekly for the presence or absence of branchial xenomas using the methodology described by Speare et al. (1998b). Fish were anaesthetized using 60 mg/L benzocaine. Using a dissecting microscope, all visible gill lamellae were examined. By week 5 post secondary exposure (week 15 in total), most of the fish housed had the presence of xenomas and were killed using an overdose of benzocaine (100 mg/L), after which the first left gill arch was dissected free and whole mounted. Xenomas were counted using a light microscope and a xenoma index (median number of xenomas per gill arch) was calculated.

3.3.6 Data Analysis

The Kruskal-Wallis, non-parametric test was applied to test the null hypothesis that the median xenoma counts per gill arch were not systematically higher in some populations. This was followed by a Bonferroni multiple comparisons test. In addition, using Chi-square analysis, significance of fish lacking xenomas in treatment groups were tested against the naive controls.

All tests were employed to compare treatments against controls. All statistical analyses were carried out using commercial software (MINITAB™ Inc., Version 15, Pennsylvania, US).

Differences were considered significant at the a=0.0125 level of probability to account for multiple comparisons within the Bonferroni tests. All other differences were considered significant at the a<0.05 level of probability.

The formula used for percent reduction of xenomas was the following:

(XCPGAcontrol - XCPGAtreatment / XCPGAcontroD * 100%

3.4 RESULTS

The Kruskal-Wallis test indicated differences among the median xenoma counts per gill arch (XCPGA) within the populations of the trial (p<0.001). Following up with Bonferroni’s

80 multiple comparisons test revealed the median xenoma counts for the groups including, high dose recovered fish, low dose recovered fish, G. anomala vaccinated and G. hertwigi vaccinated were

all significantly lower than the count for the control group (p<0.001). The total proportion of fish

in each group that showed xenomas by week 5 were 0%, 42.1%, 71.4% and 75.9%, respectively,

as outlined in Table 3.1. Analysis by Chi-square tests confirmed that the proportion of fish with

xenomas was significantly different (p<0.001) in high dose recovered fish and low dose recovered fish as compared to naive controls, whereas the G. hertwigi vaccinated and G. anomala vaccinated groups were not significantly different from naive controls (p=0.139 and p =0.238),

respectively. The high dose and low dose recovered groups were able to reduce the xenoma burden by 100% and 94.5% as compared to the control group. Although the proportion of fish with xenomas in the vaccinate groups was not significantly different from the control group, they were able to reduce the burden by 80.7% in G. anomala vaccinates and by 91.2% in G. hertwigi

vaccinates as compared to the control group.

Analysis by the Chi-square test confirmed that the XCPGA for the high dose group was

significantly different for that of the control group (p<0.001)

Mean xenoma burden was calculated for Table 3.1 by using the following equation, where 16 represents the number of hemibranchs within the branchial basket of the fish.

Mean XCPGA = ((Total xenomas/16)/n)

81 Table 3.1: Xenoma outcome at week 5, following experimental challenge with Loma salmonae, in naive vs. vaccinated fish or recovered fish.

Number % Health Total Fish % Fish Mean Reduced Status Xenom as n Infected Infected XCPGA X enom as St. Dev. Naive 20160 32 28 87.5 39.4 0 40.16 HD Rec 0 19 0 .0 0 100 0 LD Rec 656 19 8 42.1 2.2 94.5 4.03 G. hertwigi 1600 29 22 75.9 3.4 91.2 5.37 G. anomala 3392 28 20 71.4 7.6 80.7 9.96

Legend: Fish status denotes exposures prior to experimental challenge. HD Rec (recovered from high dose challenge with L. salmonae), LD Rec (recovered from low dose challenge with L. salmonae). G. hertwigi and G. anomala (indicates that these groups received a vaccination with spores from these microsporidian species). Total xenomas denotes the sum of xenomas from fish in a group (limited to xenomas counted from a hemibranch from the first branchial arch), n = number of fish per group, Mean XCPGA = ((Total xenomas/16)/n),% reduced xenomas = ((XCPGAcontrol - XCPGAtreatment)/(XCPGAcontrol))* 100%, St. Dev. = standard deviation.

82 3.5 DISCUSSION

This is the first study to demonstrate the use of novel heterologous(G. anomala and G. hertwigi) vaccines injected intraperitoneally capable of significantly reducing the xenoma burden against a high dose oral exposure of the microsporidianL. salmonae. Recent studies on the phylogeny and morphologyG. of hertwigi from rainbow smelt found in Prince Edward Island have shown that its ribosomal DNA sequence is very closely related to that ofG. anomala.

Further support thatG. hertwigi and G. anomala may be the same species is the successful experimental infection of sticklebacks with G. hertwigi from the smelt (Lovy, Koska, Dykova,

Arsenault, Peckova, Wright & Speare 2009). Based on our results, injection with spores from either Glugea species produced similar trends when it came to the percent of fish infected, the mean xenoma count per gill arch and the percent reduction of xenoma burden. Actual immune responses in fish to G. hertwigi and G. anomala, which may show intra-species differences were not investigated in the study, and it is with the collective findings on survival that indicated a close relationship withL. salmonae.

In previous work, Rodriguez-Tovar et al. (2006a) found that rainbow trout that received

IP vaccination of an inactivated semi-purified L. salmonae spore preparation 6 weeks prior to challenge at a dose of 106 were able to confer significant levels of protection against an oral challenge with L. salmonae as compared to naive controls. Since species ofLoma and those of

Glugea are phylogenetically related, it was assumed the best starting point for the study would be to adopt the exact dose rate and induction time. Although demonstrating the ability to significantly reduce xenoma burden, the vaccination of fish withG. hertwigi and G. anomala were unable to significantly reduce the proportion of fish infected with MGDS. Further investigation into not only the dose delivered, but of potential shortened or lengthened induction time should be considered.

83 Although it is labour intensive and stressful to the fish (Bruno et al. 1996), IP injections are quite common in commercial fish aquaculture and many studies have shown this route of administration is more efficacious than bath immersions or intramuscular injections (Scott 1993;

Bruno et al. 1996). A study comparing the effects of bath immersion versus IP injection of the bacterium Edwardsiella tarda, the causative agent of edwardsiellosis in turbot ( Scophthalmus maximus), demonstrated than IP injections had a relative percentage survival (RPS) rate of greater than 90%, while those fish that received bath immersion exhibited a RPS of only 13.33% (Castro,

Toranzo, Nuflez & Margariflos 2008).

Heterologous vaccines in aquaculture have been studied for many years and some studies have demonstrated the benefits of cross reactivity between species. In a laboratory study, juvenile channel catfish, Ictalurus punctatus that received IP injections of three different ciliate strains of Tetrahymena pyriformis demonstrated various significant levels of cross protection upon exposure to infectiousI. multifiliis parasites as compared to the control group in a laboratory

study (Dickerson, Brown, Dawe & Gratzek 1984). Commonly known as “Ich”, these parasites, much like L. salmonae do not grow well in culture, thereby limiting antigen availability for homologous vaccine production (Eckless & Matthews 1993). Since T. pyriformis is easily grown in culture, as reviewed by Sauvant, Pepin and Piccinni (1999), it allows for potential large-scale antigen production as compared toI. multifiliis. This production and vaccination scenario is much like that of the present study withGlugea spp. andLoma spp. Furthermore, their findings on the cross-protective potential between species correlates with this study onL. salmonae, in that some phylogenetically unrelated organisms have the ability to produce immunity against heterologous challenges (Norqvist, Hagstrom & Wolf-Watz 1989).

The current study demonstrated the reproducibility of the results from Chapter 2, as rainbow trout that have recovered from a “low dose” cohabitation infection are able to mount significant protective responses upon oral re-exposure L.to salmonae. Additionally, it further

84 validates the findings of Speare et al. (1998b), that an initial high dose exposure allows for full protection upon recovery against the onset of MGDS. The question remains as to why Chinook salmon in netpens are succumbing to this disease, assuming that these fish would certainly be living amongst infected pen mates or feral fish entering the netpen or travelling near the farm site.

With the parasite’s ability to remain latent in cool waters for many years, and changing currents with the tides, it is safe to assume that although the farmed Chinook would not necessarily be exposed to high levels of infective spores, they most certainly must be vulnerable to low levels.

It is with this daunting fact that alternative treatment approaches were investigated, and revealed promising results. The vaccination of rainbow trout with a live Glugea-spore preparation was demonstrated as allowing for partial resistance to infection with MGDS was a valuable finding, however, it is not a feasible practice in a farm scenario. Live vaccines come with varying degrees of risk, such as clinical signs or mortality directly related to the vaccine components. Although rainbow trout in the study showed no clinical signs of infections withG. hertwigi or G. anomala, it has been found that Glugea plecoglossi can successfully infect rainbow trout (Lee et al. 2004).

Since the commercialization of fresh, virulent whole spore vaccines is typically considered unfavourable due to the possibilities of disease transmission to farmed animals as well as wild fish, the idea of using freeze-thawed inactivated spores is an important consideration. Not only is this a safer method of vaccination by decreasing disease transmission, it is much more desirable commercially by having longer lasting shelf and storage life than fresh vaccine preparations.

From the encouraging results gained from the utilization of fresh spore preparations, further studies will include material which has undergone a period of inactivation to compare protection capacity.

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90 4. RESISTANCE TO AN INFECTION WITH LOMA SALMONAE IN RAINBOW TROUT (ONCORHYNCHUS MYKISS) VACCINATED WITH HETEROLOGOUS AND HOMOLOGOUS KILLED SPORES AND WITH or WITHOUT SUBSEQUENT ORAL ADMINISTRATION OF PROVALE™, A 0-1,3/1,6 GLUCAN

4.1 ABSTRACT

An in vivo challenge study (Study 1) was conducted to determine whether vaccination with killed

spores from the microsporidiansG. hertwigi, G. anomala, or a low-virulence strain of L. salmonae (SV), would protect rainbow trout from challenge with a virulent form of L. salmonae.

In a separate investigation, (Study 2) it was examined whether an immunostimulant (0-1,3/1,6 glucan) might augment immunity toL. salmonae acquired in rainbow trout which had recovered

from a low dose challenge with L. salmonae. The vaccination study, (Study 1) employed 4

groups of fish that included: a naive unvaccinated group (positive control), a group which

received a single injection with killed (inactivated) G. hertwigi spores (G. hertwigi vaccinated), a

group which received a single injection with killed (inactivated) G. anomala spores (G. anomala

vaccinated) and a group which received a single injection with killed (inactivated) L. salmonae

(SV) spores (L. salmonae SV vaccinated). Six weeks following vaccination, all fish were orally challenged with a virulent strain of L. salmonae; 5 weeks later the fish were euthanized and the

infection status (based on presence or absence of xenomas), and mean numbers of xenomas per gill arch (XCPGA) were determined and compared to the unvaccinated control group. When compared to the naive unvaccinated controls, which had an XCPGA of 72.8, all vaccinated groups were able to significantly reduce the mean number of xenomas per gill arch by 72.4% (G. hertwigi), 81.5% (G. anomala) and 66.6% (L. salmonae SV) as compared to the control group.

The second study employed 5 groups of rainbow trout which included: a naive untreated group

(positive control), a group which had recovered from a prior high dose initial challenge withL. salmonae (high dose recovered), a group which recovered from a prior low dose initial challenge with L. salmonae (low dose recovered), a group that received a 0-1,3/1,6 glucan via dietary

91 treatment (0-glucan fed) and a group that had recovered from a prior low dose initial challenge and received 0-1,3/1,6 glucan via dietary treatment (0-glucan fed + low dose recovered). When compared to the naive untreated controls, which had an XCPGA o f285.2, all groups were able to reduce the mean number of xenomas by 100%, 95%, 73.8% and 100%, respectively. Compared to the control group, all of these differences were statistically significant, however, the addition of the 0-1,3/1,6 glucan appears to have provided no additional benefit to the protective capacity of the low dose exposure group against re-challenge. This was unexpected and is likely related to the initial low level of infection received via cohabitation in the low dose groups which provided nearly full protection even without the 0-1,3/1,6 glucan. Consequently, the possibility for a commercially licensed vaccine or a dietary treatment paired with varying levels of low dose recovery againstL. salmonae should be examined more closely. Based on the striking reduction

in xenoma burden in both killed, whole spore vaccinated and immunostimulant treated groups, this justifies further work in both areas.

4.2 INTRODUCTION

Gill disease associated with the microsporidian parasite,Loma salmonae is a significant problem known to affected pen-reared salmonids, particularly Chinook salmon(O. tshawystcha)

(Speare et al. 1998c; Sanchez, Speare & Markham 1999). Experimentally rainbow trout(O. mykiss) are a suitable model for the study of disease pathogenesis, as microsporidial gill disease of salmon (MGDS) progresses similarly to that observed in Pacific salmon, by allowing completion of the parasite’s life cycle (Speare et al. 1998a). The organism has a predilection site for the endothelial and pillar cells of the gills and to a lesser extent in other vascularized tissues, such as the heart, spleen and kidney (Hauck 1984; Kent et al. 1989). Disease onset is marked by the initial formation of xenomas, which typically is evident by week 5 post exposure to the parasitic spores, with clinical signs appearing by week 7-8 post exposure, when the spore-filled xenomas rupture (Speare et al. 1998a). The resulting pathophysiological events include severe

92 branchitis, attributable to gill damage that instigates a decreased surface area for oxygen and

electrolyte exchange, as well as osmotic balance, resulting in fish vulnerability to secondary

infections and even death (Speare et al. 1989).

Once considered a significant emerging pathogen in aquaculture, the microsporidian,L. salmonae (Rodriguez-Tovar, Speare, Markham & Daley 2004, Becker & Speare 2007) is now

characterized as causing major endemics in the marine aquaculture production of salmonids

(Sanchez et al. 2001; Becker et al. 2007), distributed throughout the Pacific Northwest (Hauck

1984; Kent et al. 1989), eastern United States (Markey, Blazer, Ewing & Kocan 1994) and

Scotland (Bruno et al. 1995). Despite the levels of morbidity and mortality associated with this disease, the use of chemotherapeutants and the development of commercially available vaccines have been limited (Kent et al. 1994; Mullins, Powell, Speare & Cawthom 1994; Sanchez et al.

2001c). It is believed that the most effective vaccination method against L. salmonae is the

administration of whole spores, targeting a variety of signaling mechanisms, as opposed to

delivering single antigens which may be insufficient at targeting suitable epitopes in order to generate a sufficient protective response (Rodriguez-Tovar et al. 2006a,b). Since identification of the antigens involved and the mechanism of protection evoked is in its infancy, further investigation is required to determine the exact nature in which a resistant state is elicited.

Experimental studies using killed vaccines have revealed their efficacy against various disease agents in both aquaculture and agriculture (Xu, Shi, Shen, Lin, Wang, Lin, Qian, Ye, Fu,

Shi, Wu, Zhang, Zhu & Guo 1993; Bruno et al. 1996; Vendrell, Balcazar, Ruiz-Zarzuela, de Bias,

Giron6s & Muzquiz 2007). Xu et al. (1993) determined that an intradermal injection with freeze- killed parasites of Schistomiasis japonica was able to reduce worm burden by up to 57% in cattle when challenged percutaneously with 500 cercariae. Similarly, investigations into vaccine potential for salmonids affected byL. salmonae has determined that intraperitoneal injections with killed (inactivated) spores at a dose rate of lxlO6 spores ml"1 sterile distilled water conferred

93 between 95-100% protection 6 weeks post vaccination against an oral exposure to the parasite

(Speare et al. 1998b; Rodriguez-Tovar et al. 2006a; Speare et al. 2007). Previous findings indicate that immunity againstL. salmonae is cell-mediated, as L. salmonae is an intracellular parasite (Shaw et al. 1999), and that upon xenoma rupture, affected regions demonstrate an influx of mononuclear cells (Sanchez et al. 2001c; Speare et al. 1989; Kent et al. 1995). Furthermore,

MGDS is a prolonged disease known to cause granulomas, while exposure and recovery from the parasite allows for the induction of a strong protective response (Speare et al. 1998a,b,c; Kent et al. 1999), likely not attributable to that of humoral immunity, as the administration of serum from recovered fish to naive individuals is incapable of generating any protection (Sanchez et al.

2001c).

Immunostimulants, particularly p-1,3/1,6 glucans, are known to stimulate non-specific and specific immunity in fish. The benefits of immunostimulants have been demonstrated in a variety of species in aquaculture including Atlantic salmon (Jorgensen & Robertsen 1995), Tiger

Prawn (Panaeus monodon) (Chang, Su, Chen, Lo, Kou & Liao 1999), Pink snapperPagus ( auratus) (Cook et al. 2001), Rohu ( Labia rohita) (Sahoo & Mukheijee 2001), Asian catfish

(Clarias batrachus), (Kumari & Sahoo 2006a,b), and rainbow trout (Sakai 1999; Guselle et al.

2006). Representing ancient cell wall components, P-1,3/1,6 glucans are polysaccharides to which higher taxonomic organisms have evolved a number of recognition mechanisms, representing evolutionarily conserved innate immune responses against these cell wall derivatives

(Soltanian, Stuyven, Cox, Sorgeloos & Bossier 2009). Generally regarded as safe, while lacking toxicity and side effects, it is believed that the immune cells being stimulated by these immunoactivators are primarily macrophages (Jorgensen et al. 1995; Sahoo et al. 2001; Kumari et al. 2006a). It is the conserved microbial components of P~1,3/1,6 glucans that are primarily recognized by Dectin-1, a lectin family receptor for P-glucans found on macrophages, dendritic cells and monocytes that cooperates with Toll-like receptors (TLRs), particularly TLR2 found on macrophages and dendritic cells. This recognition begins a signaling cascade resulting in the

94 production of the cytokines, interleukin 12 (IL-12) and tumor necrosis factor a (TNF-a), which initiate a Thl-type response (Gantner, Simmons, Canavera, Akira & Underhill 2003).

Experimental studies to date have exhibited increased protective capacity of p-1,3/1,6 glucans by intraperitoneal injections than by any other means (Robertsen 1999), but like most methods of injection, it is known to cause increased cortisol levels in fish as a result of situational stress, thereby having a negative effect on the immune system and predisposing fish to secondary infections (Pickering & Pottinger 1985,1989). As compared to direct injection, reduced efficacy occurs with oral delivery by means of feed incorporation, due to the poor digestibility of P-1,3/1,6 glucans (Kumari et al. 2006a). Oral delivery is compounded by the varying dosage levels fish may receive due to tank dynamics such as hierarchy or palatability of the top coated feed.

Incorporating immunostimulants into feed rather than delivering injectable vaccines would be beneficial to marine aquaculture as it would eliminate any handling related stress that could predispose fish to disease. P* 1,3/1,6 glucans have previously been demonstrated to increase oxidative respiratory burst, lysozyme activity (Kumari et al. 2006a) and phagocytic activity

(Sahoo et al. 2001), while significantly reducing mortality associated withAeromonas hydrophila in Asian catfish, (C. batrachus) (Kumari et al. 2006a,b) and Rohu, (L. rohita) (Sahoo et al. 2001).

Intraperitoneal administration of P-1,3/1,6 glucans to rainbow trout 3 weeks prior to infection with L. salmonae was shown to be more effective at reducing the mean number of xenomas per gill arch (>95%) than any other drug tested (Guselle et al. 2006). The efficacy of P-1,3/1,6 glucans at reducing the number of xenomas is believed to be related to the function of both the site and the stage of the parasite at the time of application as timing of administration is critical.

Without a commercially licensed vaccine or ample choice when it comes to chemotherapeutants, it is unlikely that salmonid farming will ever be entirely free from the risk of

L. salmonae, as it is a water-borne pathogen, spreading with relative ease among fish (Saunders

1995). It is only with time and further experimental studies that farmers will be able to apply prophylactic treatments in order to minimize the effects of this protistan parasite. To date, there

95 has been limited success on both the vaccination against L. salmonae, as well as the oral treatment with immunostimulants against the disease. Hypotheses that might aid in the application of both a vaccine and an immunostimulant against MGDS are that heterologous killed vaccine preparations ofGlugea spp. and a homologous vaccine preparation ofLoma sp. spores delivered via intraperitoneal injection is incapable of inducing cross-protective effects upon parasite introduction. Furthermore, the oral delivery of 0-1,3/1,6 glucan will not induce any protective effects when delivered 3 weeks prior and 2 weeks post infection with L. salmonae.

These hypotheses were tested using a surrogate species(O. mykiss), undergoing a high dose challenge after vaccination and/or oral treatment with an immunostimulant in order to determine their level of protection relative to naive controls.

43 MATERIALS AND METHODS

Two trials with the following objectives were designed to evaluate the protective effects that various treatments have on the exposure toL. salmonae in rainbow trout:

Trial 1: To determine the level of cross-protection induced from receiving intraperitoneal injections of killed (inactivated) G. anomala, G. hertwigi or L. salmonae (SV) spores.

Trial 2: To re-investigate the level of protection against L. salmonae in rainbow trout that previously received oral treatment with 0-1,3/1,6 glucans and to determine if it can act synergistically with low dose recovered status to enhance resistance to challenge to virulentL. salmonae.

43.1 Sample Population

Juvenile rainbow trout weighing 30-40 grams were purchased from a certified disease- free (notifiable pathogens) commercial hatchery on Prince Edward Island, with no previous

96 history ofL. salmonae. All procedures were conducted in accordance with the guidelines of the

Canadian Council on Animal Care (CCAC 2005).

4 J.2 Experimental Design

Trial 1: Ninety rainbow trout were selected and housed in three 70 L, flow-through, circular fiberglass tanks that were supplied from a well water source, maintained at 15.0° C (±0.3°

C). The tanks had constant aeration with flow rates maintained at 9 Lmin1. After a 2 week period of acclimatization, the rainbow trout were tagged and vaccinated with either a killed preparation ofGlugea spp. orL. salmonae (SV). To maximize vaccines potentials, six weeks was allotted prior to challenge with spores ofL. salmonae.

Glugea Vaccine Preparation and Delivery

Sixty rainbow trout were selected and randomly assigned in equal groups to two 70 L tanks and allowed 2 weeks to acclimatize prior to vaccination. Xenomas of G. hertwigi were harvested from wild caught smelts, Osmerus mordex, inhabiting an estuary located in Eastern

Prince Edward Island. Spores were isolated, purified (Appendix B), underwent freeze-thaw

(Appendix C) and were administered by intraperitoneal (IP) injection to a single tank of 30 fish at a dose of 106 spores/fish in sterile saline. A second tank of 30 fish were vaccinated via IP injection at a dose rate of 106 with sterile saline and G. anomala spores that were isolated, purified (Appendix B), underwent freeze-thaw (Appendix C) from xenomas harvested from three- spined sticklebacks, Gasterosteus aculeatus and nine-spined sticklebacks, Pungitius pungitius that were housed at the Atlantic Veterinary College, Prince Edward Island. Both species of sticklebacks were kindly donated by the Biology Department at the University of Prince Edward

Island. The rainbow trout were maintained 6 weeks post exposure and monitored for changes in health. Fish returned to normal feeding behaviour 48 hours post vaccination. Three fish receiving Glugea spp. died during the lead up to challenge, for reasons unassociated with vaccination.

97 Loma Vaccine Preparation and Delivery

Thirty rainbow trout were selected and randomly assigned in to a 70 L tank and allowed 2 weeks to acclimatize prior to vaccination. Xenomas of L. salmonae (SV) were harvested from

infected Brook trout (Salvelinus fontinalis), held in the aquatic laboratory. Spores were isolated, purified, underwent freeze-thaw (Appendix D) and were administered by IP injection to a single tank of 30 fish at a dose of 106 in sterile saline. The rainbow trout were maintained 6 weeks post exposure and monitored for changes in health. Fish returned to normal feeding behaviour 48 hours post vaccination. Two fish receiving L. salmonae (SV) died during the lead up to challenge, for reasons unassociated with vaccination.

Challenge Exposure

Prior to challenge, the eighty-five fish (29G. hertwigi vaccinated, 28 G. anomala vaccinated and 28 L. salmonae (SV) vaccinated) remaining in the study were assigned to a 250 L tank at which time 34 naive rainbow trout from the same fish source were added to serve as positive controls. This trial was carried out over a period of 15 weeks.

Trial 2:

Low Dose Primary Exposure

Sixty juvenile rainbow trout were randomly selected and housed in a 250 L, flow through, circular fiberglass tank that was supplied from a well water source, maintained at 15.0°

C (±0.3° C). After a 2 week period of acclimatization, the rainbow trout were tagged and challenged by the addition of 6 rainbow trout serving as donor fish. Beginning 4 weeks post exposure, fish were screened and sampled once per week for 4 weeks. At this time all fish were considered positive for infection by visually detecting xenomas on the first left gill arch. The

98 infected fish were screened until the gills no longer showed physical signs of xenomas, at which time these fish were considered to have recovered from a primary low dose cohabitation exposure to L. salmonae. At this time the population was randomly divided in half and housed in separate

70 L tanks with water parameters as previously described. One tank was designated as “Low

Dose Recovered” and the other tank as “Low Dose Recovered + Oral ProVale™ p-glucan”.

High Dose Primary Exposure

Thirty na'fve, juvenile rainbow trout were randomly selected and housed in a 70 L tank with the same water parameters as previously described for the “Low Dose Primary Exposure.”

After a 2 week period of acclimatization, naiVe individuals received macerated gill material heavily infected with virulentL. salmonae from fish that were categorized as highly infected via microscopic examinations that were humanely euthanized by means of benzocaine overdose

(lOOmg/L). The finely minced material was diluted with tank water and introduced to the tank after a 2-day fast. All fish were feeding at the time of exposure and water flow was turned off for

1 hour to ensure contact of the parasite with the fish. Screening was done until the gills no longer showed physical signs of xenomas, demonstrating a recovery from a primary infection L.with salmonae. These fish were now deemed “High Dose Recovered.”

OralProValem 0-1,3/1,6glucan treatment

Thirty randomly selected naive rainbow trout were housed in a 70 L flow through, fiberglass tank that received oral treatments at 1% body weight with P-1,3/1,6 glucan by

(Appendix E), 3 weeks prior and 2 weeks post exposure to virulentL. salmonae (N. Guselle

Atlantic Veterinary College 2009, personal communication). These fish were housed separately from all other fish in the trial until the completion of top-coated feed treatment, at which time they were combined with the other groups.

99 Low Dose Recovered + Oral ProVale1" fl-1,3/1,6 glucan

Thirty fish were randomly selected from the pool of sixty “Low Dose Recovered” fish and housed in a 70L flow through, fiberglass tank and received oral treatments at 1% body weight with a P-1,3/1,6 glucan by (Appendix E), 3 weeks prior and 2 weeks post re-exposureL. to salmonae (N. Guselle Atlantic Veterinary College 2009, personal communication). These fish were housed separately from all other fish in the trial until the completion of feed top-coat treatment, at which time they were combined with the other groups.

Re-challenge Exposure

Prior to challenge, the “Low Dose Recovered” and “High Dose Recovered” groups were pooled into a 2S0L tank with the addition of 31 nai've rainbow trout from the same fish source to serve as positive controls. Two additional tanks were used to house and challenge the “Oral

ProVale™ p-1,3/1,6 glucan” and “Low Dose Recovered + Oral ProVale™ P-1,3/1,6 glucan” groups. After a period of 5 weeks of being housed separately, these two groups were added to the

250L experimental tank. This trial was carried out over a period of 15 weeks.

4.3.3 Method of Infection

Six weeks prior to the experimental challenge days for both trial 1 and 2, separate populations of fish were infected with macerated gill material infected withL. salmonae and once they displayed numerous mature xenomas they were euthanized by overdose with 100 mg/L benzocaine. Gills from the euthanized fish were dissected free, finely minced, diluted with tank water and introduced to the experimental tank after a 2-day fast to serve as infective material as described by Kent et al. (1995). All material was used immediately following harvest. The water flow to the tanks receiving infected gill material was turned off for 1 hour to enhance contact of the parasites with the fish (Kent et al. 1995).

100 4.3.4 Challenge Dose Quantification

Trial 1: The first left gill arch for all fish was non-lethally examined under a dissecting scope for branchial xenomas. Upon assumption of peak infection time all fish were euthanized by benzocaine overdose (100 mg/L) and the first left branchial gill arch was dissected free for whole mount and xenomas were counted using a stereomicroscope resulting in a xenoma count per gill arch (XCPGA). The estimated total weight of macerated gill material used in this trial was 13.2 grams with an estimate of49,520 xenomas delivered to the tank or roughly 412 xenomas per fish.

Trial 2: Using the same technique described for Trial 1, the total estimated weight of macerated gill material used in this trial was 10.1 grams with an estimate of 38,020 xenomas delivered to the tank or roughly 328 xenomas per fish.

4 .3.5 Sampling and Infection Assessment

Trial 1: During the trial, fish were assessed weekly for the presence or absence of branchial xenomas using the methodology described by Speare et al. (1998a). Fish were anaesthetized using 60 mg/L benzocaine. Using a dissecting microscope, all visible gill lamellae were examined. By week 5 post secondary exposure (week 15 in total), some of the fish housed had xenomas present and were euthanized with benzocaine (100 mg/L), after which the first left gill arch was dissected free and whole mounted. Xenomas were counted using a light microscope and a xenoma index (median number of xenomas per gill arch) was calculated.

Trial 2: Assessing the fish weekly as described for Trial 1, by week 5 post secondary exposure (week 15 in total), some of the fish housed had xenomas present and were euthanized by benzocaine overdose (lOOmg/L), after which the first left gill arch was dissected free and whole mounted. Xenoma counts were performed using a light microscope and a xenoma index

(mean number of xenomas per gill arch) was calculated.

101 4.3.6 Data Analysis

Trial 1: A One-way analysis of variance (ANOVA) was used to test the null hypothesis that a vaccination with Glugea spp. orLoma spp. would not confer any protection against a high dose oral exposure to macerated gill material infected withL. salmonae. Prior to administering the test, the data was natural log transformed to meet the model’s assumptions of normality

(Moore & McCabe 2006). Following the ANOVA, two-sample T-tests were then employed to reveal which treatments were significantly different from the control group.

Trial 2: Xenoma counts of zero were analyzed separately from non-zero data by testing the difference in proportions of the control and treatment groups by means of Chi-square and proportion analyses. A one-way ANOVA was used to test the null hypothesis that oral administration of 0-1,3/1,6 glucan alone and in synergy with low dose recovery fromL. salmonae would not confer any protection against a high dose oral re-exposure model. Prior to administering the test, the non-zero data was natural log transformed to meet the model’s assumptions of normality (Moore et al. 2006). Following the ANOVA, two-sample T-tests were then employed to reveal which treatments were significantly different from the control group.

All tests compared treatments against controls and all statistical analyses were carried out using commercial software (MINITAB™ Inc., Version 15, Pennsylvania, US). Differences were considered significant at the a<0.05 level of probability.

4.4 RESULTS

Trial 1: Upon analysis of the One-way ANOVA a P-value indicative of significance

(p<0.001) was found, thereby rejecting the null hypothesis and demonstrating that a vaccination with Glugea spp. orL. salmonae (SV) can confer a level of protection against a high dose oral exposure toL. salmonae. The two-sample T-tests employed for the three different vaccinations revealed that an intraperitoneal injection with Glugea hertwigi, Glugea anomala or L. salmonae

102 (SV), are all significantly different from the positive controls (p<0.001). Figure 4.1 shows that each vaccine was able to reduce the mean xenoma burden per gill arch by 72.7%, 81.5% and

66.6%, respectively, as compared to the positive control group (Table 4.1)

Trial 2: Chi-square and proportion analysis exhibited significant (p<0.001) differences between control and treatment groups in all but one, that being the groups receiving dietary treatment with 0-1,3/1,6 glucan (p=0.475). This allows for the acceptance of the null hypothesis that the oral administration of this immunostimulant alone significantly reduces xenoma burden.

Table 4.2 shows that 44.8%, 0%, 96.8% and 0% of the fish in the low dose recovered group, high dose recovered group, 0-1,3/1,6 glucan diet alone group and low dose recovered in combined with dietary 0-1,3/1,6 glucan group exhibited xenomas by week 5 post exposure respectively. All groups were able to reduce the mean xenoma burden when compared to the positive control by

99.3%, 100%, 73.8%, and 100% respectively (Table 4.2).

Analysis of the One-way ANOVA exhibited a significant P-value (p<0.001), allowing for the rejection of the null hypothesis and proving that oral administration of 0-1,3/1,6 glucan in synergy with low dose recovery fromL. salmonae confers protection against a high dose oral re­ exposure model. Following the ANOVA, two-sample T-tests were employed for each treatment with both oral administration of 0-1,3/1,6 glucan in combination with low dose recovery and low dose recovery alone revealed that both conferred significant protection (p<0.001) against a high dose challenge with L. salmonae.

103 Table 4.1: Xenoma outcome at week 5, following and experimental challenge with virulent Loma salmonae, in naive vs. vaccinated Rainbow trout.

Number % Total Fish % Fish Mean Reduced Fish Status Xenom as n Infected Infected XCPGA Xenomas St. Dev.

Trial 1

Naive 34928 30 30 100 72.8 0 60.5

G. hertwigi 9216 29 25 86.2 19.9 72.7 25.6

G. anomala 6028 28 26 92.9 13.5 81.5 13.9

L. salmonae (SV) 10880 28 27 96.4 24.3 66.6 41.4

Legend: Fish status denotes exposures prior to experimental challenge.G. hertwigi, G. anomala, and L. salmonae (SV) (indicates that these groups received a vaccination with killed spores from these microsporidian species). Total xenomas denotes the sum of xenomas from fish in a group (limited to xenomas counted from a hemibranch from the first branchial arch), n = number of fish per group, Mean XCPGA = (Total xenomas/16)/n, % reduced xenomas = ((XCPGA control - XCPGA treatment)/(XCPGA control))* 100%, St. Dev. = standard deviation.

104 Table 4.2: Xenoma outcome at week 5, following an experimental challenge with virulent Loma salmonae, in naive vs. low dose recovered, high dose recovered, P-1,3/1,6 glucan fed and p- 1,3/1,6 glucan fed in synergy with low dose recovery Rainbow trout.

Number % Total Fish % Fish Mean Reduced Fish Status Xenom as n Infected Infected XCPGA Xenomas St. Dev.

Trial 2

Naive 141440 31 31 100 285.2 0 277.6

LD Rec 992 29 13 44.8 2.1 99.3 3.7

HD Rec 0 28 0 0 0 100 0 p-glucan Fed 37024 31 30 96.8 74.6 73.8 120.9

p-glucan + LD 0 31 0 0 0 100 0

Legend: Fish status denotes exposures prior to experimental challenge. HD Rec (recovered from high dose challenge with L. salmonae), LD Rec (recovered from low dose challenge with L. salmonae), p-1,3/1,6 glucan fed (received oral treatment with immunostimulant), P-1,3/1,6 glucan fed + LD (received oral treatment with immunostimulant in synergy with recovery from low dose challenge with L. salmonae). Total xenomas denotes the sum of xenomas from fish in a group (limited to xenomas counted from a hemibranch from the first branchial arch), n = number of fish per group, Mean XCPGA = (Total xenomas/16)/n, % reduced xenomas = ((XCPGA control - XCPGA treatment)/(XCPGA control))* 100%, St. Dev. = standard deviation.

105 4.5 DISCUSSION

This is the first study to demonstrate that the use of novel killed (inactivated) heterologous vaccines injected IP against the microsporidian L. salmonae can stimulate protective responses in trout, thereby significantly reducing the xenoma burden per gill arch upon exposure to a high dose challenge. In previous studies Rodriguez-Tovar et al. (2006a) and Speare et al.

(2007) were able to confirm that the IP delivery of killed spores of L. salmonae was able to confer adequate levels of protection against re-challenge after an induction time of 6 weeks.

Building on these findings is the use of heterologous vaccines that could be used in a commercial setting, and adopting the induction time found,G. hertwigi, G. anomala and L. salmonae (SV) vaccines showed promise.

Furthermore, this is also the first study to investigate the level of protection demonstrated upon oral re-challenge of the parasite when low dose recovered fish underwent a 5 week period of

dietary treatment with the immunoactivator ProVale™, a p-1,3/1,6—glucan manufactured by

Stirling Products Ltd. Initial studies by Guselle et al. (2006) demonstrated that IP administration of p-1,3/1,6 glucans prior to experimental challenge withL. salmonae was able to reduce the xenoma burden of previously unexposed rainbow trout by up to 95% compared to control fish.

Although complete protection was achieved in fish that received synergistic treatments of P-

1,3/1,6 glucan and recovery from a low dose exposure, the findings in the laboratory study may be slightly skewed, as the low dose recovered group unexpectedly demonstrated high levels of protection (99.3% reduction of xenoma burden) compared to previous studies in Chapter 3 which demonstrated reduced xenoma burden by 94.5%. Although the difference is not statistically significant between chapters, it is difficult to distinguish whether the addition of oral treatment with the immunostimulant actually had much of an effect on the full protection against re­ challenge with L. salmonae or if this group of low dose recovered fish were able to mount the protection based on the previous infection. Further investigation into this possibility is required

106 in order to fully substantiate this hypothesis and may entail manipulating the levels of infection

introduced to naive fish by means of addition of infected cohorts.

The preparation of the killedGlugea spp. andLoma sp. vaccines were completed in a dry

lab, under optimum conditions. Previously harvested materials that was stored in the freezer at a

temperature of -20°C were used in this experiment. At the time of thawing, both theGlugea spp.

spores had been frozen for a period of 3 months while theLoma sp. spores had been frozen for 1

month under the same conditions. Once thawed, the spores were suspended in 5 ml of 0.85%

sterile saline. After centrifuging, the pelleted spore material was resuspended and a 1:10 dilution

was used to determine spore concentration using a hemocytometer. In previous studies by

Rodriguez-Tovar et al. (2006a), freeze-thawed spores have been shown to lack infectivity, and the

spores used in the study were assumed to be as well, as the time spent in the freezer was the same

as Rodriguez et al. (2006a). A dose rate of 106 was used with a period of 6 weeks post

vaccination was also employed in the study, based on favourable results in previous experimental

analysis (Rodriguez-Tovar et al. 2006a; Speare et al. 2007).

A major finding of the previous study is the demonstration that both of the unadjuvanted

heterologous vaccines were able to further decrease the xenoma burden in rainbow trout than the

widely published unadjuvanted homologous vaccine (Speare et al. 2007) by upwards of 15%. The

current study re-iterated that at a dose rate of 106 spores ofL. salmonae (SV)/100pl of sterile

saline (unadjuvanted), is able to reduce xenoma burden by about 66-69%, 6 weeks post

vaccination, indicating the reproducibility of the experiment performed by Speare et al. (2007).

The persuasive results yielded from the use of heterologous vaccines against infection withL. salmonae together with the ability to grow Glugea sp. in cell culture and the relative ease of

harvesting their xenomas in comparison toLoma sp. xenomas, suggest that the development and

commercialization of a heterologous vaccine against MGDS is a realistic perception.

107 Additionally, the results confirm previous studies by showing that fish vaccination againstL. salmonae must precede anticipated exposure to the parasite.

Adjuvants are typically incorporated into inactivated vaccines to prolong their effects and help stimulate an immune response to specific antigens (Marciani 2003; Janeway, Travers,

Walport & Shlomchik 2005). Speare et al. (2007) investigated the use of Freund’s incomplete adjuvant (FIA) added to a whole-spore low virulence strain ofL. salmonae and compared the results with a non-adjuvanted vaccine of the same strain. No significant difference was found in the ability of the adjuvanted vaccine to further decrease xenoma burden per gill arch than that of the non-adjuvanted version at dose rates between 104-106 spores/lOOpl of sterile saline (Speare et al. 2007). Clearly more studies are required to establish if unadjuvanted vaccines against L. salmonae are able to confer full protective capacity against infection.

In a similar vaccine trial, Harbour, Every, Edwards and Sutton (2008) used mice to demonstrate the protective effects that a systemic vaccination with formalin-fixed (killed)

Helicobacter pylori, delivered subcutaneously, was able to induce a protective response against both a homologous and heterologous strain via oral challenge, by significantly reducing bacteria colonization along the digestive tract. Additionally, the efficacy of the unadjuvanted whole spore vaccine was compared with that of an adjuvanted vaccine, with results indicating that neither was significantly superior at mounting a protective response in the mice upon homologous or heterologous exposure (Harbour et al. 2008). These findings indicate that some whole-spore unadjuvanted vaccines are equally efficacious as those that are adjuvanted.

Although the results yielded from the heterologous vaccination are highly persuasive, there are many outstanding questions regarding vaccination against MGDS. Does timing of exposure toL. salmonae change the protection levels of vaccinated fish? Do the fish require less, more or the same vaccine induction time as those vaccinated with homologous vaccines? Is

108 timing of fish vaccination linked to protective capacity? Answering these questions and reproducing the results obtained in the present study will bring the aspiration for a commercial vaccine against MGDS closer to reality.

109 4.6 References

BECKER J & SPEARE DJ. Transmission of the microsporidian gill parasite,Loma salmonae. Animal Health Research Reviews 2007; 8: 1-10.

BRUNO DW, COLLINS RO & MORRISON CM. The Occurrence of Loma salmonae (Protozoa: Microspora) in Fanned Rainbow Trout, Oncorhynchus mykiss Walbaum, in Scotland. Aquaculture 1995; 133: 341-44.

BRUNO DW & ELLIS AE. Salmonid Disease Management. In: Pennell W & Barton BA, eds. Principles of salmonid culture, vol. 29. Amsterdam: Elsevier Science, 1996: 759-832.

CCAC (Canadian Council on Animal Care) Guidelines on: the care and use of fish in research, teaching and testing. Ottawa: 2005.

CHANG CF, SU MS, CHEN HY, LO CF, KOU GH, LIAO IC. Effect of dietary p-l,3-glucan on resistance to white spot syndrome virus (WSSV) in postlarval and juvenilePanaeus monodon. Diseases of Aquatic Organisms 1999; 36: 163-68.

COOK MT, HAYBALL PJ, HUTCHINSON W, NOWAK B & HAYBALL JD. The efficacy of a commercial P-glucan preparation, EcoActiva™, on stimulating respiratory burst activity of head-kidney macrophages from pink snapperPagus ( auratus), Sparidae. Fish & Shellfish Immunology 2001; 11: 661-72.

GANTNER BN, SIMMONS RM, CANAVERA SJ, AKIRA S & UNDERHILL DM. Collaborative induction of inflammatory responses by Dectin-1 and Toll-like receptor 2. Journal of Experimental Medicine 2003; 197:1107-17.

GUSELLE NJ, MARKHAM RJF & SPEARE DJ. Intraperitoneal administration of p-1,3/1,6 glucan to rainbow trout, Oncorhynchus mykiss (Walbaum), protects against Loma salmonae. Journal of Fish Diseases 2006; 29: 375-81.

HARBOUR SN, EVERY AL, EDWARDS S & SUTTON P. Systemic immunization with unadjuvanted whole Heliobacter pylori protects mice against heterologous challenge. Heliobacter 2008; 13: 494-9.

HAUCK AK. A mortality and associated tissue reactions of chinook salmon,Oncorhynchus tshawytscha (Walbaum), caused by the microsporidianLoma sp. Journal of Fish Diseases 1984; 7: 217-29.

JANEWAY CA, TRAVERS P, WALPORT M & SHLOMCHIK MJ. Basic concepts in immunology. In: Janeway et al., eds. Immunobiology. New York: Garland Science, 2005:1-35.

JORGENSEN JB & ROBERTSEN B. Yeast P-glucan stimulates respiratory burst activity of Atlantic salmon (Salmo salar L.) macrophages. Developmental and Comparative Immunology 1995; 19: 43-57.

110 KENT ML & DA WE SC. Efficacy of Fumagillin DCH against experimentally inducedLoma salmonae (Microsporea) infections in chinook salmon Oncorhynchus tshawytscha. Diseases of Aquatic Organisms 1994; 20: 231-33.

KENT ML, DAWE SC & SPEARE DJ. Resistance to reinfection in chinook salmon Oncorhynchus tshawytscha to Loma salmonae (Microsporidia). Diseases of Aquatic Organisms 1999; 37: 205-08.

KENT ML, DAWE SC & SPEARE DJ. Transmission of Loma salmonae (Microsporea) to chinook salmon in sea water. Canadian Veterinary Journal 1995; 36: 98-101.

KENT ML, ELLIOT DG, GROFF JM & HEDRICK RP. Loma salmonae (Protozoa: Microspora) infections in seawater reared coho salmon Oncorhynchus kisutch. Aquaculture 1989; 80:211-22.

KUMARI J & SAHOO PK. Dietary P-1,3 glucan potentiates innate immunity and disease resistance of Asian catfish, Clarias batrachus (L.). Journal of Fish Diseases 2006a; 29: 95-101.

KUMARI J & SAHOO PK. Dietary immunostimulants influence specific immune response and resistance of healthy and immunocompromised Asian catfishClarias batrachus to Aeromonas hydrophila infection. Diseases of Aquatic Organisms 2006b; 70: 63-70.

MARCIANI DJ. Vaccine adjuvants: role and mechanisms of action in vaccine immunogenicity. Drug Discovery Today 2003; 8: 934-43.

MARKEY PT, BLAZER VS, EWING MS & KOCAN KM. Loma sp. in Salmonids from the Eastern United States: Associated Lesions in Rainbow Trout. Journal of Aquatic Animal Health 1994; 6: 318-28.

MOORE DS & MCCABE GP. Two-Way Analysis of Variance. Introduction to the Practice of Statistics. 5th Edition. W.H. Freeman and Company: New York, 2006: 771-88.

MULLINS JE, POWELL MJ, SPEARE DJ & CAWTHORN R. An intranuclear microsporidian in lumpfish Cyclopterus lumpus. Diseases of Aquatic Organisms 1994; 20: 7-13.

PICKERING AD & POTTINGER TG. Cortisol can increase the susceptibility of brown trout, Salmo trutta L., to disease without reducing the white blood cell count. Journal of Fish Biology 1985;27:611-19.

PICKERING AD & POTTINGER TG. Stress responses and disease resistance in salmonid fish: effects of chronic elevation of plasma cortisol. Fish Physiology and Biochemistry 1989; 7: 253- 58.

ROBERTSEN B. Modulation of non-specific defense of fish by structurally conserved microbial polymers. Fish & Shellfish Immunology 1999; 9: 269-90.

RODRIGUEZ-TOVAR LE, BECKER JA, MARKHAM RJF & SPEARE DJ. Induction time for resistance to microsporidial gill disease caused byLoma salmonae vaccination of rainbow trout

111 (Oncorhynchus mykiss) with a spore-based vaccine. Fish & Shellfish Immunology 2006a; 21: 170-75.

RODRIGUEZ-TOVAR LE, MARKHAM RJF, SPEARE DJ & SHEPPARD J. Cellular immunity in salmonids infected with the microsporidial parasiteLoma salmonae or exposed to non-viable spores. Veterinary Immunology and Immunopathology 2006b; 114: 72-83.

RODRIGUEZ-TOVAR LE, SPEARE DJ, MARKHAM RJF & DALEY J. Predictive modeling of post-onset xenoma growth during microsporidial gill disease(Loma salmonae) of salmonids. Journal of Comparative Pathology 2004; 131: 330-33.

SAHOO PK & MUKHERJEE SC. Effect of dietary 0-1,3 glucan on immune responses and disease resistance of healthy and aflatoxin Bi-induced immunocompromised rohu(Labeo rohita Hamilton). Fish & Shellfish Immunology 2001; 11: 683-95.

SAKAI M. Current research status of fish immunostimulants. Aquaculture 1999; 172: 63-92.

SANCHEZ JG, SPEARE DJ & MARKHAM RJF. Nonisotopic detection ofLoma salmonae (Microspora) in rainbow trout (Oncorhynchus mykiss) gills by in situ hybridization. Veterinary Pathology 1999; 36: 610-12.

SANCHEZ JG, SPEARE DJ, MARKHAM RJF & JONES SRM. Experimental vaccination of rainbow trout against Loma salmonae using a live low-virulence variant of Loma salmonae. Journal of Fish Biology 2001c; 59:442-48.

SANCHEZ JG, SPEARE DJ, MARKHAM RJF, WRIGHT GM & KIBENGE FSB. Localization of the initial developmental stages ofLoma salmonae in rainbow trout (Oncorhynchus mykiss). Veterinary Pathology 2001; 38: 540-46.

SAUNDERS RL. Salmon Aquaculture: Present Status and Prospects for the Future. In: Boghen AD, ed. Cold-water aquaculture in Atlantic Canada, 2nd Ed. Sackville: The Tribune Press Ltd., 1995:37-81.

SHAW RW & KENT ML. Fish Microsporidia. In: Wittner M & Weiss LM, eds. The Microsporidia and Microsporidiosis. Washington: American Society for Microbiology, 1999: 418-46.

SOLTANIAN S, STUYVEN E, COX E, SORGELOOS P & BOSSIER P. Beta-glucans as immunostimulant in vertebrates and invertebrates. Critical Reviews in Microbiology 2009; 35: 109-38.

SPEARE DJ, ARSENAULT GJ & BUOTE MA. Evaluation of rainbow trout as a model for use in studies on pathogenesis of the branchial microsporidianLoma salmonae. Contemporary Topics 1998a; 37: 55-8.

SPEARE DJ, BRACKETT J & FERGUSON HW. Sequential pathology of the gills of coho salmon with a combined diatom and microsporidian gill infection. Canadian Veterinary Journal 1989; 30: 571-75.

112 SPEARE DJ, DALEY J, MARKHAM RJF, SHEPPARD J, BEAMAN HJ & SANCHEZ JG. Loma salmonae-d&soci&XeA growth rate suppression in rainbow trout,Oncorhynchus mykiss (Walbaum), occurs during early onset xenoma distribution as determined byin situ hybridization and immunohistochemistry. Journal of Fish Diseases 1998c; 21: 345-54.

SPEARE DJ, MARKHAM RJF & GUSELLE NJ. Development of an effective whole-spore vaccine to protect against microsporidial gill disease in rainbow trout (

VENDRELL D, BALCAZAR JL, RUIZ-ZARZUELA I, DE BLAS I, GIRONES O & MUZQUIZ JL. Safety and efficacy of and inactivated vaccine againstLactococcus garvieae in rainbow trout ( Oncorhynchus mykiss). Preventative Veterinary Medicine 2007; 20; 222-29.

XU S, SHI F, SHEN W, LIN J, WANG Y, LIN B, QIAN C, YE P, FU L, SHI Y, WU W, ZHANG Z, ZHU H & GUO W. Vaccination of bovines against Schistomiasis japonica with cryopreserved-irradiated and ffeeze-thaw schistosomula. Veterinary Parasitology 1993; 47: 37- 50.

113 5. GENERAL DISCUSSION

The ability of some microsporidia to impact fish health negatively is well known and although many treatments have proven effective (Speare et al. 1998d; Sanchez et al. 2001; Becker et al. 2004; Guselle et al. 2007; Speare et al. 2007), none are commercially licensed. It has been previously shown that naturally- and vaccine-acquired immunity against other fish pathogens has led to methods of control (Hedrick et al. 1988; Bruno et al. 1996; Chang et al. 1999; Corbeil,

Lapatra, Anderson & Kurath 2000; Cook et al. 2001; Sommerset, Lorenzen, Lorenzen, Bleie &

Nerland 2003; Xu et al. 2006), including L. salmonae (Speare et al 1998a; S&nchez et al. 2001,

Guselle et al. 2007; Speare et al. 2007).

Marine netpen culture of salmonids allows fish to be exposed not only to pathogens carried by their penmates, but also those associated with the natural population of feral fish that surround and interact with the farm-reared fish (Munro 1990; Kent 2000; Hammell & Dohoo

2005). The concept of herd immunity is important in this type of aquaculture rearing practice and is recognized as a proportion of any given population having immunity to a certain pathogen

(John & Samuel 2000). This theory of herd immunity raises the question as to why salmonids in marine netpens, suspected of being exposed to low levels ofL. salmonae (Shaw et al. 1998;

Ramsay et al. 2003), continue to suffer high levels of morbidity and mortality at a mature and highly valuable stage in life (Hauck 1984; Kent et al. 1989; Markey et al. 1994; Bruno et al.

1995).

A curious finding regarding infection with L. salmonae is that an experimental low dose exposure, by means of cohabitating heavily infected donor rainbow trout with naive fish and subsequent recovery, was able to offer a moderate level of protection against a high dose oral re­ exposure model in every experimental trial. Since partial protection is observed with recovery from a low dose cohabitation exposure in the laboratory setting but is not seen in netpen

114 aquaculture, the reproducibility of this experimental finding was examined. All studies

demonstrated fish exposed to low doses had significantly reduced xenoma burden as compared to

the controls. These findings contradict what has been reported to occur in a netpen setting, as the

results suggest that herd immunity should be generated within a given population exposed to the

spores of thisL. salmonae, yet these netpen fish continue to suffer enormously when it comes to

this endemic disease.

Additionally, since the findings that vaccination with a low virulent (SV) strain ofL. salmonae (Sanchez 2001; Speare et al. 2007) was protective, the possibility for vaccination with a

heterologous microsporidian species was investigated to assess if cross-protection could be

developed against a high dose oral exposure model.

Investigation of cross-protective effects of two species of the microsporidianGlugea (G.

anomala and G. hertwigi) gave impressive results: fresh spore preparations injected IP 6 weeks prior to virulent challenge reduced the xenoma burden of rainbow trout by 80.7% and 91.2%,

compared to the control group respectively. A particular weakness in this investigation is the lack

of a fresh spore preparation ofL. salmonae (SV) for comparison purposes. This vaccine is

lacking in Chapter 3 as the aquatic facility in which the fish vaccinated with freshL. salmonae

spores were housed underwent a period in which chlorine contaminated the water system. These

fish were humanely euthanized due to animal ethics concerns and the fresh spore group was therefore lost in this particular study. As a substitute, a group of fish that recovered from a high dose oral challenge was added to the study for comparison purposes, as well as for testing the reproducibility of a previous study by Speare et al. (1998a). The results were promising, as 100% protection was achieved. Regardless of these results and the fact that fresh spore live vaccine preparations are not available for commercial licensing, further investigation to evaluate the use of freeze-thaw (inactivated) spores to confer protective responses against a challenge withL. salmonae was necessary.

115 Since 2001, IP injections of a freeze-thaw preparation ofL. salmonae SV spores at a dose

rate of 106 spores/lOOpl of sterile saline has given the best protection against oral exposure of the

microsporidian, L. salmonae after oral challenge (Sanchez et al. 2001). This may no longer be considered to be the best method of protection, as IP injections of freeze-thaw heterologous

Glugea species, (G. anomala and G. hertwigi) were shown to further reduce the XCPGA burden

as compared to inactivated homologous L. salmonae SV injections. By reducing the mean xenoma count per gill arch by 81.5%, 72.7% and 66.6%, respectively,G. anomala and G.

hertwigi were upwards of 15% more effective than L. salmonae SV. This is a major finding, as not only do inactivated vaccines have more commercial licensing potential, but the microsporidian species ofG. anomala and G. hertwigi are non-salmonid pathogens, known to

endemically infect three- and nine-spined sticklebacks, (Gasterosteus aculeatus and Pungitius pungitius) (Lorn et al. 2005) and smelt (Osmerus mordax), on the East and West coasts of Canada

and the United States (Haley 1954; Nepszy et al. 1978). Application of heterologous vaccines is

not a new practice and has been employed to control many diseases of both humans and animals

(Behbehani 1983; Norqvist et al. 1989; Corbeil et al. 2000; Clark 2001; Sommerset et al. 2003;

Xu et al. 2006; Baras, Stittelaar, Simon, Thoolen, Mossman, Pistoor, Van Amerongen,

Wettendorf, Hanon & Osterhaus 2008; Harbour et al. 2008). Currently no licensed vaccines are

available against microsporidians affecting fish (Speare et al. 1998b), a great launch point to

further investigate cross-protective potentials within species of microsporidian. The concept of

heterologous vaccines against microsporidia known to affect teleosts is a valuable probe for phylogenetically related species or those having overlapping general characteristics. It is worthy of noting that in aquatic farm settings, vaccination is not always the most practical method of

exposure as it is costly and stressful to the fish from the handling and injections (Bruno et al.

1996), making this a general weakness in the experimental aspects of the study.

116 Since vaccination is a huge undertaking in a farm situation, alternative methods to confer protection againstL. salmonae were investigated. Because partial protection is noted upon recovery from a low dose cohabitation challenge, the idea of a feed additive of an immunostimulant to increase this level of protection was explored. The immunostimulant choice was based on availability and affordability, as the product ProVale™ is manufactured on Prince

Edward Island and has given excellent results in previous studies (N. Guselle Atlantic Veterinary

College 2009, personal communication). In Chapter 4, fish that recovered from a low dose infection with L. salmonae and fed a dietary treatment with (1-1,3/1,6 glucan showed no signs of infection upon re-exposure to the parasite. Since the general understanding is that fish in a farm situation would be continually exposed to spores L.of salmonae, the use of a dietary treatment that includes an immunostimulant is favourable. Having the ability to treat numerous fish ranging in size with little to no stress involved in the treatment is ideal for this type of situation.

A drawback to oral treatment is the ability to dictate when to begin treatment because fish must be treated prophylactically 2 weeks prior to exposure and 3 weeks post exposure for the best results (N. Guselle Atlantic Veterinary College 2009, personal communication). In this type of situation, the studies completed on water temperature and the development ofL. salmonae should be consulted in order to estimate when fish in a netpen are the most likely to be exposed to parasitic spores (Beaman et al. 1999a). Additionally, the synergistic effects of recovery from a low dose exposure and immunostimulant therapy may apply broadly when it comes to parasite burdens of other species in various kinds of aquaculture. This is exemplified by the potential to reduce the epizootic “white spot” syndrome caused by a ciliated protistan,Ichthyophthirius multifiliis in channel catfish (Ictalurus punctatus), as a recovery from a cohabitation model leads to partial immunity (Xu, Klesius & Shoemaker 2007). The data obtained in this thesis research suggest that the use of immunostimulant therapy in combination with low dose exposure to various pathogens of both freshwater and seawater fish may improve the level of protection.

117 Although the findings regarding naturally- and vaccine-induced resistance are very exciting, there are still areas of research that need further investigating before implementation of these strategies or licensing any products. Reproducibility of the findings within the thesis is absolutely necessary as time constraints and animal and tank availability limited replication of experiments. Furthermore, the experimental studies explored within the thesis employed a rainbow trout model (Speare et al. 1998a), and it is therefore necessary to investigate whether the findings are applicable to Chinook salmon. Finally, investigation into the synergistic effects of not only 0-1,3/1,6 glucan but also other immunostimulants commonly used in aquaculture (Sakai

1999; Bricknell & Dalmo 2005) with IP injections of freeze-thaw preparations ofG. anomala, G. hertwigi and L. salmonae SV may reveal some valuable findings.

Diseases in an aquaculture setting are dependent upon interactions between pathogen, host and environment. All studies herein employed rainbow trout as a surrogate model when it comes to MGDS. Taking into account that this disease causes severe mortality in netpens, where salinity, host species and exposure conditions vary considerably, it is imperative that further research accounts for these differences. Additionally, further exploration of the common antigens of microsporidia may uncover valuable information that could lead to superior protection than those observed in Chapter 4. Identifying and categorizing species affording cross protection against L. salmonae is of great importance to the future control and potential eradication of

MGDS in aquaculture.

118 5.1 References

BARAS B, STITTELAAR KJ, SIMON JH, THOOLEN RJMM, MOSSMAN SP, PISTOOR FHM, VAN AMERONGEN G, WETTENDORF MA, HANON E & OSTERHAUS ADME. Cross-protection against lethal H5N1 challenge in ferrets with an adjuvanted pandemic influenza vaccine. PLoS ONE 2008; 3:1-4.

BEAMAN HJ, SPEARE DJ & BRIMACOMBE M. Regulatory effects of water temperature on Loma salmonae (Microspora) development in rainbow trout. Journal of Aquatic Animal Health 1999a; 11:237-45.

BECKER J & SPEARE DJ. Ultraviolet light control of horizontal transmission of Loma salmonae. Journal of Fish Diseases 2004; 27: 177-80.

BEHBEHANI AM. The smallpox story: life and death of an old disease. Microbiological Reviews 1983; 47: 455-509.

BRICKNELLI & DALMO RA. The use of immunostimulants in fish larval aquaculture. Fish & Shellfish Immunology 2005; 19: 457-72.

BRUNO DW, COLLINS RO & MORRISON CM. The Occurrence of Loma salmonae (Protozoa: Microspora) in Farmed Rainbow Trout, Oncorhynchus mykiss Walbaum, in Scotland. Aquaculture 1995; 133: 341-44.

BRUNO DW & ELLIS AE. Salmonid Disease Management. In: Pennell W & Barton BA, eds. Principles of salmonid culture, vol. 29. Amsterdam: Elsevier Science, 1996: 759-832.

CHANG CF, SU MS, CHEN HY, LO CF, KOU GH, LIAO IC. Effect of dietary p-l,3-glucan on resistance to white spot syndrome vims (WSSV) in postlarval and juvenilePanaeus monodon. Diseases of Aquatic Organisms 1999; 36: 163-68.

CLARK IA. Heterologous immunity revisited. Parasitology 2001; 122: S51-S59.

COOK MT, HAYBALL PJ, HUTCHINSON W, NOWAK B & HAYBALL JD. The efficacy of a commercial 0-glucan preparation, EcoActiva™, on stimulating respiratory burst activity of head-kidney macrophages from pink snapperPagus ( auratus), Sparidae. Fish & Shellfish Immunology 2001; 11: 661-72.

CORBEIL S, LAPATRA SE, ANDERSON ED & KURATH G. Nanogram quantities of a DNA vaccine protect rainbow trout fry against heterologous strains of infectious hematopoietic necrosis virus. Vaccine 2000; 18:2817-824.

GUSELLE NJ, MARKHAM RJF & SPEARE DJ. Timing of intraperitoneal administration of 0- 1,3/1,6 glucan to rainbow trout, Oncorhynchus mykiss (Walbaum), affects protection against the microsporidianLoma salmonae. Journal of Fish Diseases 2007; 30: 111-16.

119 HALEY AJ. Microsporidian parasite,Glugea hertwigi, in American smelt from the Great Bay region, New Hampshire. Transactions of the American Fisheries Society 1954; 83:84-90.

HAMMELL KL & DOHOO IR. Risk factors associated with mortalities attributed to infectious salmon anaemia virus in New Brunswick, Canada. Journal of Fish Diseases 2005; 28: 651-61.

HARBOUR SN, EVERY AL, EDWARDS S & SUTTON P. Systemic immunization with unadjuvanted whole Heliobacter pylori protects mice against heterologous challenge. Heliobacter 2008; 13:494-9.

HAUCK AK. A mortality and associated tissue reactions of chinook salmon,Oncorhynchus tshawytscha (Walbaum), caused by the microsporidianLoma sp. Journal of Fish Diseases 1984; 7: 217-29.

HEDRICK RP, GROFF JM, FOLEY P & MCDOWELL T. Oral Administration of Fumagillin DCH protects Chinook salmon Oncorhynchus tshawystcha from experimentally-induced proliferative kidney disease. Diseases of Aquatic Organisms 1988; 4: 165-68.

JOHN TJ & SAMUEL R. Herd immunity and herd effect: new insights and definitions. European Journal of Epidemiology 2000; 16: 601-6.

KENT ML. Marine netpen farming leads to infections with some unusual parasites. International Journal for Parasitology 2000; 30: 321-26.

KENT ML, ELLIOT DG, GROFF JM & HEDRICK RP. Loma salmonae (Protozoa: Microspora) infections in seawater reared coho salmon Oncorhynchus kisutch. Aquaculture 1989; 80:211-22.

LOM J & DYKOVA I. Microsporidian xenomas in fish seen in wider perspective. Folia Parasitologica 2005; 52: 69-81.

MARKEY PT, BLAZER VS, EWING MS & KOCAN KM. Loma sp. in Salmonids from the Eastern United States: Associated Lesions in Rainbow Trout. Journal of Aquatic Animal Health 1994; 6:318-28.

MUNROALS. Salmon farming. Fisheries Research 1990; 10:151-61.

RAMSAY JM, SPEARE DJ, BECKER JA, DALEY J. Loma salmonae-associatcd xenoma onset and clearance in rainbow trout, Oncorhynchus mykiss (Walbaum): comparisons ofper os and cohabitation exposure using survival analysis. Aquaculture research 2003; 34: 1329-35.

NEPSZY S, BUDD J & DECHTIAR AO. Mortality of young-of-the-year rainbow smelt (Osmerus mordax) in Lake Erie associated with the occurrence of Glugea hertwigi. Journal of Wildlife Diseases 1978; 14:233-39.

NORQVIST A, HAGSTROM A & WOLF-WATZ H. Protection of rainbow trout against vibriosis and furunculosis by the use of attenuated strains of Vibrio anguillarum. Applied and Environmental Microbiology 1989; 55: 1400-05.

120 SAKAI M. Current research status of fish immunostimulants. Aquaculture 1999; 172: 63-92.

SANCHEZ JG, SPEARE DJ, MARKHAM RJF, WRIGHT GM & KIBENGE FSB. Localization of the initial developmental stages ofLoma salmonae in rainbow trout ( Oncorhynchus mykiss). Veterinary Pathology 2001; 38: 540-46.

SHAW RW, KENT ML & ADAMSON ML. Modes of transmission of Loma salmonae (Microsporidia). Diseases of Aquatic Organisms 1998; 33: 151-56.

SOMMERSET I, LORENZEN E, LORENZEN N, BLEIE H & NERLAND AH. A DNA vaccine against a rainbow trout rhabdovirus induces early protection against a nodavirus in turbot. Vaccine 2003; 21:4661-67.

SPEARE DJ, ARSENAULT GJ & BUOTE MA. Evaluation of rainbow trout as a model for use in studies on pathogenesis of the branchial microsporidianLoma salmonae. Contemporary Topics 1998a; 37: 55-8.

SPEARE DJ, BEAMAN HJ, JONES SRM, MARKHAM RJF & ARSENAULT GJ. Induced resistance in rainbow trout, Oncorhynchus mykiss (Walbaum), to gill disease associated with the microsporidian gill parasiteLoma salmonae. Journal of Fish Diseases 1998b; 21: 93-100.

SPEARE DJ, MARKHAM RJF & GUSELLE NJ. Development of an effective whole-spore vaccine to protect against microsporidial gill disease in rainbow trout (Oncorhynchus mykiss) by using a low-virulence strain of Loma salmonae. Clinical and Vaccine Immunology 2007; 14: 1652-54.

SPEARE DJ, RITTER G & SCHMIDT H. Quinine hydrochloride treatment delays xenoma formation and dissolution in rainbow trout challenged with Loma salmonae. Journal of Comparative Pathology 1998d; 119: 459-65.

XU DH, KLESIUS PH, PANANGALA VS. Induced cross-protection in channel catfish, Ictalurus punctatus (Rafinesque), against different immobilization serotypes ofIchthyopthirius multifiliis. Journal of Fish Diseases 2006; 29: 131-38.

121 APPENDIX A

1. Department of Fisheries and Oceans aquaculture production and value in 2010 in Canadian dollars.

Type Quantity Value (Tonnes) (CDN dollars) Finfish 122,161 846,101,000 Shellfish 38,765 73,361,000 Total 160,925 919,462,000

2. Department of Fisheries and Oceans 2010 Canadian aquaculture production statistics

(tones)(1)

Nfld PEI NS NB Que Ont Man Sask Alta BC CANADA Finfish

Salmon{3) X X 4,960 25,625 0 0 0 0 0 70,800 101,385 Trout(3) 0 X 128 150 344 4,060 XXX 600 6,883 Steelhead(3) X 0 0 0 0 0 0 0 0 0 0

Other<4) XX 91 X ,, XXX X 600 993

Total Finfish(2) 12,889 X 5,179 25,775 344 4,060 X XX 72,000 122,161 Shellfish Clams 0 0 438 0 0 0 0 0 0 1,500 1,938 Oysters 0 2,377 205 881 0 0 0 0 0 7,400 10,862 Mussels<3) 2,461 18,845 2,121 95 563 0 0 0 0 400 24,484 Scallops(3) 0 0 2 0 0 0 0 0 0 700 702 Other 0 0 172 0 5 0 0 0 0 600 777 Total Shellfish 2,461 21,2212,939 976 568 0 0 0 0 10,600 38,765 Total Aquaculture(Z> 15,360 21321 8,11726,751 912 4,060 21 XX 82,600 160,924 Re-stocking(1) 401 401 Total (incL Re- stocking)(,) 15360 21321 8,11726,751 1313 4,060 21 XX 82,600 161326

(1) To outfitters: operations offering lodging and services for hunting, fishing and trapping. (2> Excludes other finfish for all provinces (3> Excludes confidential data at Canada level. (4) Other finfish data is only available at the Canada level and includes confidential data from the provinces.

122 APPENDIX B

Protocol for freshGlugea spp. Spore Purification Technique:

1. Dissect xenomas from various body tissues

2. Place intact xenomas into centrifuge tube

3. Mechanically rupture xenomas and suspend in 5 ml of 0.85% sterile saline

4. Centrifuge at 1,000 rpm for 10 minutes at 4°C

5. Resuspend the pellet in 5 ml of 0.85% sterile saline

6. Use a 1:10 dilution of this to count the spores with the haemocytometer

123 APPENDIX C

Protocol for freeze-thaw (inactivated)Glugea spp. Spore Purification Technique:

1. Dissect xenomas from various body tissues

2. Freeze intact xenomas for 2-4 weeks at -20°C

3. Thaw frozen material at room temperature

4. Suspend material in 5 ml of 0.85% sterile saline

5. Centrifuge at 1,000 rpm for 10 minutes at 4°C

6. Resuspend the pellet in 5 ml of 0.85% sterile saline

7. Use a 1:10 dilution of this to count the spores with the haemocytometer

V

124 APPENDIX D

Protocol for freeze-thaw (inactivated)L. salmonae SV Spore Purification Technique:

1. Remove gills from fish

2. Remove cartilage from gills

3. Chop gills using a razor blade

4. Grind macerated gills using a tissue grinder (Wheaton)

5. Suspend in 0.85% sterile saline

6. Centrifuge at 1,000 ipm for 10 minutes at 4°C

7. Pour off supernatant and pass pelleted material through a Cellector screen using 0.85%

sterile saline

8. Pass resulting material through Nytex mesh (50 pm) using 0.85% sterile saline

9. Centrifuge at 1,000 rpm for 10 minutes at 4°C

10. Repeat steps 8 and 9

11. Discard supernatant

12. Add 6% (v/v) Triton-X 100 to pelletized material and vortex for 30 seconds

13. Centrifuge at 2,000 rpm for 15 minutes at 4°C

14. Pour off supernatant

15. Resuspend in 0.85% sterile saline

16. Repeat step 13

17. Resuspend pelletized material in 4 mL 0.85% sterile saline and 2% (v/v)

Penicillin/Streptomycin stock solution

18. Count spores using haemocytometer at 40X magnification

19. Store solution at -20°C for 4 weeks

20. Thaw solution and inject into fish at a dose rate of 106

125 APPENDIX E

Protocol for the preparation of P-1,3/1,6 glucan top coated feed: 1. Boil 100 ml of distilled water 2. Weigh 4 grams of Oxoid™ (Oxoid Ltd., Basingstoke, Hampshire, England) gelatin powder 3. Add 70 ml of boiled distilled water to gelatin powder in a clean bowl 4. Allow gelatin mixture to cool 5. Weigh 0.1 grams of 0-1,3/1,6 glucan ProVale™ (Stirling, Charlottetown, Prince Edward Island, Canada) 6. Add measured 0-1,3/1,6 glucan ProVale™ to cooled gelatin mixture and stir 7. Weigh 500 grams of 3mm pelletized Corey™ (Corey Feed Mills Ltd., Fredericton, New Brunswick, Canada) feed 8. Pour p-1,3/1,6 glucan ProVale™ and gelatin mixture onto feed and stir until food is evenly coated 9. Place coated feed in a single layer onto a baking sheet and allow to air dry 10. Store feed in air tight containers in fridge

126