COMPARISON OF NATURALLY-ACQUIRED AND … · JENNIFER E. HARKNESS A Thesis ... SIGNATURE PAGE AV/J*...
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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
©APRIL 2012. J.E. HARKNESS
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whole or in part should be addressed to:
Chair of the Department of Pathology and Microbiology
Faculty of Veterinary Medicine
University of Prince Edward Island
Charlottetown, P. E. I.
Canada CIA 4P3
ABSTRACT
Microsporidial Gill Disease of Salmon (MGDS) is a result of infection with Loma 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 control L. 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 to L. 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 to L. 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 to L. salmonae, the intraperitoneal (IP) injection into RBT 6 weeks preexposure to L. 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 to L. 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.
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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
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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 INTRODUCTION1.1 Aquaculture 1
1.1.1 Global Production 11.1.2 Canadian Production 21.1.3 Salmonid Production 41.1.4 Chinook Salmon 51.1.5 Netpens 7
1.2 Disease in Netpens 91.2.1 Diseases of Chinook Salmon 101.2.2 Protistan Diseases of Salmon in Seawater 11
1.3 Microsporidian Parasites 131.3.1 Microsporidian Life Cycle 151.3.2 Microsporidians in Human and Animal Health 18
1.4 Loma salmonae 201.4.1 Transmission 211.4.2 Diagnosis and Clinical Signs 221.4.3 Immunological Resistance 261.4.4 Drug Treatment 291.4.5 Vaccines Against Loma salmonae 32
1.5 Research Objectives 331.6 REFERENCES 35
2. COMPARATIVE RESISTANCE TO LOMA SALMONAE IN RAINBOW TROUT (ONCORHYNCHUS MYKISS) SUBSEQUENT TO RECOVERY FROM AN INITIAL LOW DOSE COHABITATION EXPOSURE2.1 ABSTRACT 482.2 INTRODUCTION 49
2.3 MATERIALS AND METHODS 522.3.1 Sample Population 522.3.2 Experimental Design 522.3.3 Methods of Infection 532.3.4 Challenge Dose Quantification 532.3.5 Sampling and Infection Assessment 532.3.6 Data Analysis 54
2.4 RESULTS 552.5 DISCUSSION 582.6 REFERENCES 64
FIRST DEMONSTRATION OF THE PARTIAL EFFICACY OF AHETEROLOGOUS VACCINE AGAINST LOMA SALMONAE, USING SPORESFROM TWO SPECIES OF GLUGEA3.1 ABSTRACT 683.2 INTRODUCTION 693.3 MATERIALS AND METHODS 76
3.3.1 Sample Population 763.3.2 Experimental Design 773.3.3 Methods of Infection 793.3.4 Challenge Dose Quantification 793.3.5 Sampling and Infection Assessment 803.3.6 Data Analysis 80
3.4 RESULTS 803.5 DISCUSSION 833.6 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 914.2 INTRODUCTION 924.3 MATERIALS AND METHODS 96
4.3.1 Sample Population 964.3.2 Experimental Design 974.3.3 Methods of Infection 1004.3.4 Challenge Dose Quantification 1014.3.5 Sampling and Infection Assessment 1014.3.6 Data Analysis 102
4.4 RESULTS 1024.5 DISCUSSION 1064.6 REFERENCES 110
GENERAL DISCUSSION 1145.1 REFERENCES 119
APPENDIX A 122APPENDIX B 123
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 ruptured Loma 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
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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.
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LIST OF ABBREVIATIONS
°c -Degrees Celsiusfil -Microlitrepm -Micrometre% -PercentAGD -Amoebic Gill DiseaseDNA -Deoxyribonucleic acidDCH -DicyclohexamineFIA -Freund's incomplete adjuvantFig. -FigureERM -Enteric Red Mouthg -GramsHPAI -Highly Pathogenic Avian Influenza VirusHsp70 -Heat shock protein 70i.e. -Id est; that isIHNV -Infectious Hematopoeitic Necrosis VirusIL-12 -Interleukin 12IP -IntraperitonealKBMA -Killed but metabolically activekg -KilogramsL -Litrelbs -Poundsm -MetreMb -Megabasemg -MilligramsMGDS -Microsporidial Gill Disease of Salmonmin -Minutesml -MillilitresMNC -Mononuclear cellsOA -Oncorhynchus associatedP -P-valuePA -Protective antigenPE -Post exposurePCR -Polymerase chain reactionrDNA -Ribosomal deoxyribonucleic acidRNA -Ribonucleic AcidRPS -Relative percentage survivalSGR -Specific growth rateSV -Salvelinus variantTLRs -Toll-like receptorsTNF-a -Tumor necrosis factor av/v -Volume for volumeXCPGA -Xenoma count per gill arch
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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
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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 o f46,000 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).
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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
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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
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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).
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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 2 1 -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).
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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).
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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 include Nucleospora (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 whereas K. 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 genera Encephalitozoon, 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 understood in vivo, experimental studies in 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 stages (Glugea 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 including Encephalitozoon, 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 parasite Glugea 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 genus Loma 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) reported L. 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 of L. 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 ruptured Loma 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 against L. 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 where L. 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 of L. 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 of L. salmonae is able to render the parasite significantly less
virulent to naive fish than those being delivered unaltered.
The discovery of two variants of L. 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 to L.
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 of L. salmonae is not limited to Oncorhynchus spp. but
extends also to the Salvelinus 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 with L. 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 to L. 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 to L. 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 to L. 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 to L. salmonae. Compared to chemotherapeutants
experimentally used to control L. 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 of L. 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 with L. 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 on L. salmonae were investigated
by Speare et al. (1998d). Commonly used as a treatment for malaria (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 against L. 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 against L. 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-exposure to L. 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 ferret (Mustela 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 against L. 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 to L. salmonae, and this was used to answer the
following questions:
1. What is the relative protection against a high dose oral exposure to L. 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 against L. 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 against L. salmonae?
34
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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 by Loma
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 exposed to L. 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 with L. 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.
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2.2 INTRODUCTION
Since its recognition in 1988 (Speare et al. 1989), MGDS caused by the obligate
intracellular pathogen L. salmonae (Lom 2002), has become an endemic seasonal cause of
morbidity and mortality affecting populations of Chinook salmon (Oncorhynchus 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, including L. 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 as Ichthyophthirius 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 to L. 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 with L. 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 to L. 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 with L. 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
23 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 of L. 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 with L. 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 with L. 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 to L. 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.
2 3 5 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 reported L. 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
TankHealthStatus n
NumberFish
Infected°/o Fish
InfectedMean
XCPGA
%ReducedX enom as St.D ev.
A Naive 20 19 95 44.5 0 42.8LD Rec 38 21 55 8.1 89.4 6.9
B Naive 20 20 100 34.4 0 61.8LD 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 to L. salmonae are capable of mounting an overall significant protective
response against re-exposure to the parasite of L. 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. Further L. 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 previous in 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 of L. 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 to L. 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 for L. 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 of L.
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 with L.
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 microsporidian L. 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 with L. 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 the L. 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 of L. salmonae being encountered
by farmed salmon at this latter part of their production cycle.
63
2.6 References
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BAXA-ANTONIO D, GROFF JM & HEDRICK RP. Experimental Horizontal Transmission of Enterocytozoon salmonis to Chinook Salmon, Oncorhyrtchus tshawytscha. Journal of Protozoology 1992; 39: 699-702.
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.
BECKER JA, SPEARE DJ & DOHOOIR. Effect of the number of infected fish and acute exposure period on the horizontal transmission of Loma salmonae (Microsporidia) in rainbow trout, Oncorhynchus mykiss. Aquaculture 2005; 244: 1-9.
BECKER JA, SPEARE DJ & DOHOO IR. Effect of water temperature and flow rate on the transmission of microsporidial gill disease caused by Loma salmonae in rainbow trout Oncorhynchus mykiss. The Japenese Society of Fish Pathology 2003; 38:105-112.
BOUWKNEGT M, RUTJES SA, REUSKEN CB, STOCKHOFE-ZURWIEDEN N, FRANKENA K, DE JONG MC, DE RODA HUSMAN AM & POEL WH. The course of hepatitis E virus infection in pigs after contact-infection and intravenous inoculation. BMC Veterinary Research 2009; 5:1-12.
BRANSON E. Environmental aspects of aquaculture. In: Brown L, eds. Aquaculture for Veterinarians: Fish Husbandry and Medicine. Oxford: Pergamon Press, 1993: 57-67.
BRETT JR. Environmental factors and growth. In: Hoar WS, Randall DJ & Brett JR, eds. Fish physiology: bioenergetics and growth, Vol. 8. New York: Academic Press, Inc., 1979: 599-675.
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COLLINS R. Principles of disease diagnosis. In: Brown L, eds. Aquaculture for Veterinarians: Fish Husbandry and Medicine. Oxford: Pergamon Press, 1993: 69-90.
FINDLAY VL, HELDERS M, MUNDAY BL & GURNEY R. Demonstration of resistance to reinfection with Paramoeba sp. by Atlantic salmon, Salmo salar L. Journal of Fish Diseases 1995; 18: 639-42.
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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.
JOHNSON SC. Crustacean Parasites. In: Kent ML & Poppe TT, eds. Diseases of Seawater Netpen-Reared Salmonid Fishes. British Columbia: Pacific Biological Station, 1998: 80-90.
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.
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.
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.
RAMSAY JM, SPEARE DJ, BECKER JA, DALEY J. Loma salmonae-associated xenoma onset and clearance in rainbow trout, Oncorhynchus mykiss (Walbaum): comparisons of per 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 by Loma salmonae vaccination of rainbow trout (iOncorhynchus mykiss) with a spore-based vaccine. Fish & Shellfish Immunology 2006a; 21: 170-75.
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RODRIGUEZ-TOVAR LE, MARKHAM RJF, SPEARE DJ & SHEPPARD J. Cellular immunity in salmonids infected with the microsporidial parasite Loma 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 of Loma 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 of Loma 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 microsporidian Loma salmonae. Contemporary Topics 1998a; 37: 55-8.
SPEARE DJ, BEAMAN HJ & DALEY J. Effect of water temperature manipulation on a thermal unit predictive model for Loma 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 parasite Loma 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 by in 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
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using a low-virulence strain of Loma salmonae. Clinical and Vaccine Immunology 2007; 14: 1652-54.
STOSKOPF M. Anaesthesia. In: Brown L, eds. Aquaculture for Veterinarians: Fish Husbandry and Medicine. Oxford: Pergamon Press, 1993: 161-67.
TRAXLER GS, KENT ML & POPPE TT. Viral Diseases. In: Kent ML & Poppe TT, eds. Diseases of Seawater Netpen-Reared Salmonid Fishes. British Columbia: Pacific Biological Station, 1998: 36-48.
XU DH, KLESIUS PH, PANANGALA VS. Induced cross-protection in channel catfish, Ictalurus punctatus (Rafinesque), against different immobilization serotypes of Ichthyopthirius multifiliis. Journal of Fish Diseases 2006; 29: 131-38.
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3. FIRST DEMONSTRATION OF THE PARTIAL EFFICACY OF AHETEROLOGOUS 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 with L. 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 to Loma (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 against L. 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 to L.
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 of Nosema 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 against L. 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 against Encephalitozoon 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 to E. 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 to L. 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 of L. 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, growing Loma 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 speciesft
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 of Glugea to Loma would suggest that spores from
species in this genus conceivably could generate cross protection. Both Loma 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 to L. 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 genus Glugea
are therefore readily available to study the potential of cross-protection against infection with L.
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 two Glugea 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.
33 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 with L. salmonae. The number of donor fish used for cohabitation
was based on their infection status and previous studies on the transmission potential of L.
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 with L. 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 with L. 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 with L.
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.
HealthStatus
TotalXenom as n
NumberFish
Infected% Fish
InfectedMean
XCPGA
%ReducedX enom as St. Dev.
Naive 20160 32 28 87.5 39.4 0 40 .16HD Rec 0 19 0 .0 0 100 0LD Rec 656 19 8 42.1 2.2 94.5 4 .03
G. hertwigi 1600 29 22 75.9 3.4 91.2 5.37G. 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 microsporidian L. salmonae. Recent studies on the
phylogeny and morphology of G. hertwigi from rainbow smelt found in Prince Edward Island
have shown that its ribosomal DNA sequence is very closely related to that of G. anomala.
Further support that G. 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 with L. 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 of Loma 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 with G. 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 infectious I. 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 to I. multifiliis. This production and vaccination scenario is
much like that of the present study with Glugea spp. and Loma spp. Furthermore, their findings
on the cross-protective potential between species correlates with this study on L. 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 to L. 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 with G. 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.
85
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EDLIND TD, LI J, VISVESVARA GS, VODKIN MH, MCLAUGHLIN GL & KATIYAR SK. Phylogenetic analysis of p-tubulin sequences from amitochondrial protozoa. Molecular Phylogenetics and Evolution 1996; 5: 359-67.
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LEE S-J, YOKOYAMA H & OGAWA K. Modes of transmission of Glugea plecoglossi (Microspora) via the skin and digestive tract in an experimental infection model using rainbow trout, Oncorhynchus mykiss (Walbaum). Journal o f Fish Diseases 2004; 27: 435-44.
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4. RESISTANCE TO AN INFECTION WITH LOMA SALMONAE IN RAINBOWTROUT (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 microsporidians G. 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 to L. 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 with L.
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
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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 against L. 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
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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 by L. salmonae has determined that intraperitoneal injections
with killed (inactivated) spores at a dose rate of lxlO6 spores ml"1 sterile distilled water conferred
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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 against L. 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 snapper (Pagus
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
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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 with Aeromonas 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
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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 of Glugea spp. and a homologous vaccine preparation of Loma 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 to L. 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 virulent L.
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
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history of L. 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 of Glugea spp. or L. salmonae (SV). To maximize vaccines potentials, six weeks was
allotted prior to challenge with spores of L. 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.
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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 (29 G. 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
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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 Pro Vale™ 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 virulent L. 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 with L.
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 virulent L. 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.
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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-exposure to L.
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 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 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 o f49,520 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. or Loma spp. would not confer any protection against a high
dose oral exposure to macerated gill material infected with L. 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 from L. 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. or L. salmonae (SV) can confer a level of protection against a high dose oral
exposure to L. 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 from L. 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.
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Table 4.1: Xenoma outcome at week 5, following and experimental challenge with virulent Loma salmonae, in naive vs. vaccinated Rainbow trout.
Fish StatusTotal
Xenom as n
NumberFish
Infected% Fish
InfectedMean
XCPGA
%ReducedX enom as 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.
Fish StatusTotal
Xenom as n
NumberFish
Infected% Fish
InfectedMean
XCPGA
%ReducedX enom as 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 with L. 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 killed Glugea spp. and Loma 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 the Glugea spp.
spores had been frozen for a period of 3 months while the Loma 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 of L. 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 with L.
salmonae together with the ability to grow Glugea sp. in cell culture and the relative ease of
harvesting their xenomas in comparison to Loma 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 against L.
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 of L. 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 to L. 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
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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 juvenile Panaeus 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 snapper (Pagus 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 microsporidian Loma 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.
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KENT ML & DA WE SC. Efficacy of Fumagillin DCH against experimentally induced Loma 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 catfish Clarias 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 by Loma salmonae vaccination of rainbow trout
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(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 parasite Loma 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 of Loma 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 of Loma 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.
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SPEARE DJ, ARSENAULT GJ & BUOTE MA. Evaluation of rainbow trout as a model for use in studies on pathogenesis of the branchial microsporidian Loma salmonae. Contemporary Topics 1998a; 37: 55-8.
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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 by in 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 (<Oncorhynchus mykiss) by using a low-virulence strain of Loma salmonae. Clinical and Vaccine Immunology 2007; 14: 1652-54.
VENDRELL D, BALCAZAR JL, RUIZ-ZARZUELA I, DE BLAS I, GIRONES O & MUZQUIZ JL. Safety and efficacy of and inactivated vaccine against Lactococcus 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.
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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 of L. 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 this L. 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 of L.
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 microsporidian Glugea (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 of L. salmonae (SV) for comparison purposes. This vaccine is
lacking in Chapter 3 as the aquatic facility in which the fish vaccinated with fresh L. 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 with L.
salmonae was necessary.
115
Since 2001, IP injections of a freeze-thaw preparation of L. 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 of G. 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 against L. 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 of L. 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 of L. 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 of G. 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
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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 snapper (Pagus 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 microsporidian Loma 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.
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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 of per 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.
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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 of Loma salmonae in rainbow trout (Oncorhynchus mykiss). Veterinary Pathology 2001; 38: 540-46.
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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.
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APPENDIX A
1. Department of Fisheries and Oceans aquaculture production and value in 2010 in Canadian dollars.
Type Quantity Value(Tonnes) (CDN dollars)
Finfish 122,161 846,101,000Shellfish 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 CANADAFinfishSalmon{3) X X 4,960 25,625 0 0 0 0 0 70,800 101,385Trout(3) 0 X 128 150 344 4,060 X X X 600 6,883Steelhead(3) X 0 0 0 0 0 0 0 0 0 0Other<4) X X 91 X ,, X X X X 600 993Total Finfish(2) 12,889 X 5,179 25,775 344 4,060 X X X 72,000 122,161ShellfishClams 0 0 438 0 0 0 0 0 0 1,500 1,938Oysters 0 2,377 205 881 0 0 0 0 0 7,400 10,862Mussels<3) 2,461 18,845 2,121 95 563 0 0 0 0 400 24,484Scallops(3) 0 0 2 0 0 0 0 0 0 700 702Other 0 0 172 0 5 0 0 0 0 600 777Total Shellfish 2,461 21,221 2,939 976 568 0 0 0 0 10,600 38,765Total Aquaculture(Z> 15,360 21321 8,117 26,751 912 4,060 21 X X 82,600 160,924Re-stocking(1) Total (incL Re- stocking)(,) 15360 21321 8,117 26,751
401
1313 4,060 21 X X 82,600
401
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.
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APPENDIX B
Protocol for fresh Glugea 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
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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