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The biology of infection by loma salmonae (microsporidia) Shaw, Ross Winning 1999-12-31

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THE BIOLOGY OF INFECTION BY LOMA SALMONAE  (MICROSPORIDIA)  by ROSS WINNING SHAW B.Sc, The University of Alberta, 1991 M.Sc, The University of Maine, 1994 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES (Department of Zoology) We accept this thesis as conforming to the required standard  THE UNIVERSITY OF BRITISH COLUMBIA June 1999 © Ross Winning Shaw, 1999  In presenting  this  degree at the  thesis  in  partial fulfilment  of  University of  British Columbia,  I agree  freely available for reference copying  of  department  this or  publication of  and study.  his  or  her  representatives.  Department of  "2-.QQ1 Q  The University of British Columbia-' Vancouver, Canada  Date  DE-6 (2/88)  1 CjQj  that the  may be It  this thesis for financial gain shall not  permission.  requirements  I further agree  thesis for scholarly purposes by  the  is  that  an  advanced  Library shall make it  permission for extensive  granted by the understood  be  for  that  allowed without  head  of  my  copying  or  my written  ABSTRACT Loma salmonae  is an important gill pathogen of Pacific salmon  Oncorhynchus  spp. in the Pacific Northwest. Epizootics of the parasite have caused considerable economic loss in Pacific salmon farming in British Columbia. A study was undertaken on the basic biology of L. salmonae  since very little information exists to aid fish-farmers  and scientists. Loma salmonae  transmission was examined by challenging fish by per  os (oral), injection, and cohabitation with infected fish. Loma salmonae  is transmissible  by experimental (e.g. intraperitoneal, intramuscular injection) and natural (e.g.  peros,  cohabitation of naive and infected fish) exposure routes. Autoinfection of hosts is possible as demonstrated by intravascular injections of spores producing infection. The route of infection in salmon was examined using histological techniques. Sporoplasms from extruded spores were detected in epithelial cells of the alimentary canal. The parasite may move through these cells and enter the blood stream eventually reaching the gills. Purified spores were held under various laboratory conditions to examine viability. Spores were still infective after 95 d at 4 °C in fresh or sea water. Spores were killed when frozen or exposed to 100 or 200 ppm iodophor, although some spores survived the latter treatment. Host specificity of L. salmonae  was examined by per os  exposure of salmonids and nonsalmonds. All Oncorhynchus  spp. tested were  susceptible, but Atlantic salmon and Arctic char were resistant. All nonsalmonids tested were resistant. A Northern Stream strain of Chinook showed higher susceptibility to the parasite when compared to a Southern Coastal or hybrid strains. Macrophage phagocytic ability was investigated as a possible mechanism to explain differences between strains and susceptible or resistant salmonids. No differences were found between strains but Atlantic salmon macrophages have a higher phagocytic response to  L. salmonae  spores than Chinook. The possibility of nonsalmonid reservoirs for L. ii  salmonae  was examined by collecting different life stages of salmonids and  nonsalmonids near Vancouver Island. Loma salmonae  was present throughout the life  cycle of wild salmonids and a new Loma sp. is described from Cymatogaster  aggregata  using morphology, transmission studies, and rDNA sequencing.  iii  TABLE OF CONTENTS Abstract  ii  List of Tables  vi  List of Figures  viii  Preface  xiv  Acknowledgements  xv  CHAPTER I  CHAPTER II  CHAPTER III  CHAPTER IV  CHAPTER V  Overview Fish-Infecting Microsporidia  1  Introduction Immunology Treatments Significant Microsporidian Diseases Summary and Thesis Overview  1 4 9 11 37  Modes of Transmission  56  Introduction Materials and Methods Results Discussion  56 57 60 61  Viability Under Various Laboratory Conditions  72  Introduction Materials and Methods Results Discussion  72 73 75 76  Host Susceptibility  88  Introduction Materials and Methods Results  88 89 91  Discussion  91  Immunology  .'  104  Introduction  104  Materials and Methods Results Discussion  105 107 108 iv  CHAPTER VI  CHAPTER VII  Wild Salmonid and Nonsalmonid Hosts Introduction Materials and Methods Results Discussion  122 122 126 129  Conclusions and Future Recommendations  146  Bibliography Appendix I  .....122  152 Peer Reviewed Articles  175  v  TABLES  Table 1. Loma salmonae  in Oncorhynchus  tshawytscha  and O. kisutch. Infection of L.  salmonae in chinook and coho salmon exposed by various routes. Fish were examined at 56 d post-exposure and considered positive upon detection of the parasite in wet mounts or histological sections 64 Table 2. Loma  salmonae  in Oncorhynchus  kisutch.  Infection of L. salmonae  in coho  from trial 8 exposed by various routes to 1.2 x 10 spores fish" (except controls). Fish were considered positive upon detection of the parasite in wet mounts or histological sections 66 6  Table 3. Loma salmonae  in Oncorhynchus  1  kisutch. Distribution of L. salmonae  in organs  of coho salmon from trials 2 and 8 combined, at 56 d after exposure by various routes. Values for tissue are a percentage of fish showing infection in organ indicated 67 Table 4. Loma salmonae spore viability as measured by methylene blue exclusion for various treatments in dechlorinated (aquarium) fresh water in trial 79 Table 5. Percent viability of Loma salmonae spores incubated in fresh water (aquarium) or sea water (ambient) for various times as measured by methylene blue exclusion, per os infection of chinook salmon Oncorhynchus tshawytscha, and changes in spore number in trial 3 80 Table 6. Prevalence of Loma salmonae in chinook salmon Oncorhynchus tshawytscha infected per os with spores previously incubated in fresh water (aquarium), sea water (ambient), or Earl's Balanced Salt Solution (EBSS) over time for 3 separate trials 81 Table 7. Gill material infected with Loma salmonae was stored under various conditions and times before per os exposure to chinook salmon Oncorhynchus tshawytscha at 4.1 x 10 spores fish 82 6  Table 8. Prevalence and intensity of L. salmonae in chinook salmon Oncorhynchus tshawytscha infected per os by a gill homogenate previously exposed to various concentrations of iodine. Fish were examined 56 and 70 d after exposure  83  Table 9. Species of fish tested for susceptibility to Loma salmonae listed by source, and dosage of parasite. Chinook salmon Oncorhynchus tshawytscha were used as positive controls 95 Table 10. Prevalence of Loma salmonae in species tested for susceptibility by feeding infected macerated gill tissue and by peros administration of an infected gill slurry. Chinook salmon Oncorhynchus tshawytscha were used as positive controls 96 Table 11. Prevalence and intensity of Loma salmonae infection in 3 strains of chinook salmon Oncorhynchus tshawytscha after exposure by feeding infected gill tissue (trial 1) or administration peros (trial 2) of an infected gill slurry 97 vi  Table 12. Significant differences in Loma salmonae xenoma intensity between various comparisons of 3 strains of chinook salmon Oncorhynchus tshawytscha in 2 trials...98 Table 13. Average xenoma intensity in mortalities of 3 strains of chinook salmon Oncorhynchus  tshawytscha  infected by per os with Loma salmonae  in trial 2  99  Table 14. Distribution of Loma salmonae in organs of 3 strains of chinook salmon Oncorhynchus tshawytscha after exposure by feeding infected gill tissue (trial 1) or administration per os (trial 2) of an infected gill slurry. Values for tissue are percentage of fish showing infection in organ indicated 100 Table 15. Average length and weight of Atlantic Salmo sa/ar salmon and 2 strains of chinook salmon Oncorhynchus tshawytscha that macrophages were purified from for immunoflurescent (IFAT) and yeast assays 113 Table 16. Macrophage percent phagocytosis (percent of spores engulfed) of Loma salmonae spores between various comparisons of Atlantic Salmo salar salmon, and 2 strains of chinook salmon Oncorhynchus tshawytscha. Spores were preincubated in the plasma treatments 114 Table 17. Macrophage phagocytic index (average number engulfed) of Loma salmonae spores between various comparisons of Atlantic Salmo salar salmon, and 2 strains of chinook salmon Oncorhynchus tshawytscha. Spores were preincubated in the plasma treatments 115 Table 18. Infection statistics for Loma embiotocia found in Cymatogaster collected at 3 sites around Vancouver Island, British Columbia in 1995  aggregata 133  Table 19. Comparison of Loma embiotocia host, morphological, and developmental characteristics with Loma species infecting the gills of various fishes. Characteristics of turns in filament, xenoma wall, sporoblast vacuolation, and sporophorous vesicle (SPV) are ultrastructural. SPV formation is summarized as occurring before, during sporogony, or after spore formation 134 Table 20. Prevalence and intensity of Loma salmonae in 4 species of salmonid sampled from various locations on Vancouver Island, and 1 mainland location in British Columbia, Canada 136  FIGURES Fig. 1. Pleistophora Paracheirodon  hyphessobryconis  infection in skeletal muscle of neon tetra  inessi. Scale bar = 14 pm  Fig. 2. Microsporidium sp. infecting the central nervous system of zebrafish rerio. Arrows denote xenomas. Scale bar = 35 pm  38 Brachydanio  39  Fig. 3. Wet mount of a ruptured xenoma filled with spores of undescribed Loma sp. from the gill of ling cod Ophiodon elongatus. Scale bar = 12 pm 40 Fig. 4. Pleistophora hippoglossoideos in the musculature of plaice Hippoglossoides platessoides. Reprinted with permission of the author and publisher from Moller & Moller (1986)  41  Fig. 5. Heavy infection of Loma salmonae (white xenomas) in fresh gill of wild spawning sockeye salmon Oncorhynchus nerka from Babine lake, British Columbia, Canada. Reprinted with permission from M. Higgins 42 Fig. 6. Wet mount of spores from an undescribed microsporidian from mountain whitefish Prosopium williamsoni. Arrow indicates large posterior vacuole. Scale bar = 10 pm 43 Fig. 7. Electron micrograph of phagocytic cell with prominent nucleus (N) and remnants of Pleistophora ovariae spore walls within a food vacuole (FV). Scale bar = 2 pm. Reprinted with permission of the author and publisher from Canning & Lorn (1986)....44 Fig. 8. Large cysts of Glugea anomala in a stickleback Gasterosteus aculeatus Reprinted with permission of the author and publisher from Moller & Moller (1986)  45  Fig. 9. Abdominal cysts of Glugea hertwigidenoted by arrows in rainbow smelt Osmerus mordax. Reprinted with permission of the author and publisher from Noga (1996) 46 Fig. 10. Glugea stephani'm winter flounder Pleuronectes americanus. Heavy infection of cysts in the intestine. Reprinted with permission of the author and publisher from Moller & Moller (1986) 47 Fig. 11. Glugea stephani'\r\ winter flounder Pleuronectes americanus. Close up to show pebbled chalky-white appearance of intestine in the same individual. Reprinted with permission of the author and publisher from Moller & Moller (1986) 47 Fig. 12. Various stages of microsporidian development represented by histological sections of Loma salmonae in coho salmon Oncorhynchus kisutch gills. Young xenoma in secondary lamellae denoted by arrow 48 Fig. 13. Various stages of microsporidian development represented by histological sections of Loma salmonae in coho salmon Oncorhynchus kisutch gills. Mature xenoma with few remnants of host cell nuclei denoted by arrow. Scale bar = 45 pm 48 viii  Fig. 14. Various stages of microsporidian development represented by histological sections of Loma salmonae in coho salmon Oncorhynchus kisutch gills. Proliferative inflammatory response denoted by arrows associated with infection in the primary lamellae 48 Fig. 15. Various stages of microsporidian development represented by histological sections of Loma salmonae in coho salmon Oncorhynchus kisutch gills. Granuloma resulting from a ruptured xenoma with a few remaining spores denoted by arrow in macrophages. Scale bar = 10 urn 48 Fig. 16. Tetramicra Scophthalmus  brevifilum denoted by arrows in skeletal musculature of turbot maximus. Reprinted with permission of the author and publisher from  Matthews & Matthews (1980)  49  Fig. 17. Pleistophora ovariae in the golden shiner Notemigonus with permission from G. Hoffman  chrysoleucas.  Reprinted 50  Fig. 18. Sporophorocyts of Heterosporis finiki\r\ the musculature of angelfish Pterophyllum scalare. Scale bar = 20 |im. Reprinted with permission of the publisher from Michel et al. (1989) 51 Fig. 19. Micrsporidium takedailn heart of sockeye salmon Oncorhynchus nerka. Reprinted with permission of the author and publisher from Canning & Lorn (1986)..52 Fig. 20. Microsporidium takedai'm musculature of rainbow trout Oncorhynchus mykiss. Scale bar = 4 urn. Reproduced with permission of the authors and publisher from Lorn & Dykova (1992) 52 Fig. 21. Reno-splenomegaly in chinook salmon Oncorhynchus with Nucleospora salmonis. K: kidney; S: spleen  tshawytscha  infected 53  Fig. 22. Nucleospora salmonis denoted by arrows in the nuclei of hemoblasts from the retrobulbar tissue of the eye of chinook salmon Oncorhynchus tshawytscha in histological section. Scale bar = 10 urn 54 Fig. 23. Nucleospora salmonis denoted by arrows in the nuclei of hemoblasts from the retrobulbar tissue of the eye of chinook salmon Oncorhynchus tshawytscha in histological section. Gram stain of the same tissue, shows spores in nucleus remnant and free denoted by arrows. Scale bar = 10 urn 54 Fig. 24. Electron micrograph of Nucleospora Scale bar = 0.25 (im  salmonis  in the nucleus of a hemoblast , 55  Fig. 25. Loma salmonae in Oncorhynchus kisutch. Hematoxylin & eosin sections of L. spores in coho salmon infected per os and sampled within 6.5 h. Spores in stomach material denoted by arrows. Scale bar = 12 |im 68 salmonae  ix  Fig. 26. Loma salmonae  in Oncorhynchus  kisutch. Hematoxylin & eosin sections of L.  salmonae spores in coho salmon infected peros and sampled within 6.5 h. Single spore in association with stomach epithelial cells. Scale bar = 12 pm 69 Fig. 27. Loma salmonae  in Oncorhynchus  kisutch. Hematoxylin & eosin sections of  possible sporoplasms (denoted by arrows) of L. salmonae in anterior intestinal epithelial cells of coho salmon infected peros and sampled within 24 h. Sporoplasm in intestinal cell. Scale bar = 6 pm 69 Fig. 28. Loma salmonae  in Oncorhynchus  kisutch. Hematoxylin & eosin sections of  possible sporoplasms (denoted by arrows) of L. salmonae in anterior intestinal epithelial cells of coho salmon infected peros and sampled within 24 h. Sporoplasm in association with lamina propria of intestinal cells. Scale bar = 6 pm 69 Fig. 29. Loma salmonae  in Oncorhynchus  kisutch. Hematoxylin & eosin sections of  possible sporoplasms (denoted by arrows) of L. salmonae in anterior intestinal epithelial cells of coho salmon infected per os and sampled within 24 h. Sporoplasm in association with lamina propria of intestinal cells.Scale bar = 6 pm 69 Fig. 30. Loma salmonae  in Oncorhynchus  kisutch. L. salmonae  spores detected by  I FAT in the blood of coho salmon within 24 h of fish being injected with pure spores via the ventral gill sinus. Scale bar = 12 pm 70 Fig. 31. Loma salmonae  in Oncorhynchus  kisutch. Pronounced inflammatory response  in coho salmon muscle after 56 d in association with intramuscular injection of L. salmonae spores. Hematoxylin & eosin. Scale bar = 6 pm  71  Fig. 32. Viability of Loma salmonae spores held in various solutions at 10°C over time as measured by methylene blue exclusion in trial 2 84 Fig. 33. Average percent extrusion of Loma salmonae spores in a 3% H2O2 solution from trial 4-6, after storage at 4°C in fresh water (aquarium), sea water (ambient), or Earl's balanced salt solution (EBSS) overtime 85 Fig. 34. Average xenoma intensity of Loma salmonae in chinook salmon Oncorhynchus tshawytscha from trial 5 & 6 infected per os with spores stored at 4°C in fresh water (aquarium) for increasing periods of time 86 Fig. 35. Average xenoma intensity of Loma salmonae in chinook salmon Oncorhynchus tshawytscha from trials 5 & 6 infected per os with spores stored at 4°C in sea water (ambient) for increasing periods of time 87 Fig. 36. Mortality in 3 strains of chinook salmon Oncorhynchus after peros infection with Loma salmonae  tshawytscha  over time 101  Fig. 37. Histological section of a Loma salmonae xenoma denoted by arrow in a chinook salmon Oncorhynchus tshawytscha artery adjacent to pyloric caeca. Scale bar = 14 pm 102 x  Fig. 38. Histological sections of Loma salmonae xenomas in chinook salmon Oncorhynchus tshawytscha tissue. Xenoma denoted by arrow in a section of intestine. Scale bar = 20 um 103 Fig. 39. Histological sections of Loma salmonae xenomas in chinook salmon Oncorhynchus tshawytscha tissue. Xenoma denoted by arrow in dermis of dorsal muscle section. Scale bar = 20 um 103 Fig. 40. Histological sections of Loma salmonae xenomas in chinook salmon Oncorhynchus tshawytscha tissue. Xenoma denoted by arrow in mesentery. Scale bar = 20 um 103 Fig. 41. Loma salmonae  phagocytosed by macrophages of Salmo salar and  Oncorhychus tshawytscha. Immunofluorescent (IFAT) fields with green L. salmonae spores and counterstained red macrophages. Spores were preincubated in normal plasma (Atlantic salmon) or no plasma (chinook salmon) Spores phagocytosed by Atlantic macrophages denoted by arrow. Scale bar = 6 um 116 Fig. 42. Loma salmonae  phagocytosed by macrophages of Salmo salar and  Oncorhychus tshawytscha. Immunofluorescent (IFAT) fields with green L. salmonae spores and counterstained red macrophages. Spores were preincubated in normal plasma (Atlantic salmon) or no plasma (chinook salmon). Spores phagocytosed by chinook macrophages denoted by arrow and unphagocytosed denoted by arrowheads. Scale bar = 6 um 116 Fig. 43. Saccharomyces cerevisiae (arrow) phagocytosed by macrophages of Oncorhynchus tshawytscha stained with Wright's Modified Leukostat Stain. Scale bar = 6 um 117 Fig. 44. Percent of Loma salmonae spores engulfed by macrophages (percent phagocytosis) of Atlantic salmon Salmo salar, and 2 strains of chinook salmon Oncorhynchus tshawytscha. Spores were previously incubated in plasma treatments  118  Fig. 45. Percent of baker's yeast Sarccharomyces cerevisiae cells engulfed by macrophages (percent phagocytosis) of Atlantic salmon Salmo salar, and 2 strains of chinook salmon Oncorhynchus tshawytscha. Yeast cells were previously incubated in plasma treatments 119 Fig. 46. Average number of Loma salmonae spores engulfed by macrophages (phagocytic index) of Atlantic salmon Salmo salar, and 2 strains of chinook salmon Oncorhynchus tshawytscha. Spores were previously incubated in plasma treatments. Error bars represent standard deviations 120 Fig. 47. Average number of baker's yeast Sarccharomyces cerevisiae cells engulfed by macrophages (phagocytic index) of Atlantic salmon Salmo salar, and 2 strains of chinook salmon Oncorhynchus tshawytscha. Yeast cells were previously incubated in plasma treatments. Error bars represent standard deviations 121  Fig. 48. Fresh spores of Loma embiotocia um  observed under oil immersion. Scale bar = 10 137  Fig. 49. Xenoma of the hypertrophic cell type formed by Loma embiotocia associated with shiner perch Cymatogaster aggregata gill secondary lamellae. N; host cell nucleus remnants. Scale bar = 10 urn 138 Fig. 50. Transmission electron micrographs of Loma embiotocia spores and xenoma structure. Mature spore with 17 coils of the polar filament denoted by arrow. Scale bar = 1 um 139 Fig. 51. Transmission electron micrographs of Loma embiotocia spores and xenoma structure. Small tubules denoted by arrow within xenoma cytoplasm. Scale bar= 1 um 139 Fig. 52. Transmission electron micrographs of Loma embiotocia spores and xenoma structure. Sporoblasts (Sb) with minimal vacuolation within sporophorous vesicles (Spv). Scale bar = 1 um 140 Fig. 53. Transmission electron micrographs of Loma embiotocia spores and xenoma structure. Smooth xenoma wall (W) containing spores (S). Scale bar = 1 um 140 Fig. 54. PCR of DNA from chinook salmon gill infected with Loma salmonae and from gills of shiner perch infected with L. embiotocia. Shiner perch were collected at Eagle Bay, Tofino Dock, and Nanaimo sites, and parasite DNA amplified with primers Loma f and LS-2 or primers LS-1 and LS-2 (1% agarose gel, ethidium-bromide stained). Negative control used distilled water instead of template DNA. Molecular weight markers (bp) are shown in the left lane 141 Fig. 55. Sequence alignment of the gene fragments sequenced here, including a portion (334 bp) of the small subunit ribosomal DNA (SSU rDNA) and part (185 bp) of the large subunit DNA (LSU rDNA), and the internal transcribed spacer (ITS), for Loma salmonae and Loma embiotocia (GenBank U78815). Lowercase letters denote the presumptive ITS. Differences between L. salmonae and L. embiotocia are marked with "*". Differences between L. embiotocia from different localities are marked with +. R indicates both G and A, W indicates both A and T, and M indicates both C and A 142 Fig. 56. PCR of DNA from sockeye salmon Oncorhynchus nerka gill infected with Loma salmonae. Parasite DNA amplified with primers LS-1 and LS-2 (2% agarose gel, ethidium-bromide stained). The negative and positive controls (Ctr), respectively, used distilled water and DNA from purified L. salmonae spores. Lanes 5 to 16 are grouped samples of 5 fish per lane. Fish were collected at Great Central Lake, Vancouver Island, British Columbia, Canada as migrating smolts (1996 y class). Note positives in lanes 6, 8, 10, 12 & 15. Molecular weight markers (bp) are shown in lane 1 & 4 143  xii  Fig. 57. PCR of DNA from sockeye salmon Oncorhynchus nerka gill infected with Loma salmonae. Parasite DNA amplified with primers LS-1 and LS-2 (1% agarose gel, ethidium-bromide stained). The negative and positive controls (lanes 1, 35 & 2, 36) respectively, used distilled water and DNA from purified L. salmonae spores. Fish were collected at Great Central Lake, Vancouver Island, British Columbia, Canada as migrating smolts (1997 y class). Note nonspecific bands in lanes 44, 46, 48 & 57 and 1 positive in lane 58. Molecular weight markers (bp) are shown in lanes 19, 34, 37 & 54 144 Fig. 58. PCR of DNA from sockeye salmon Oncorhynchus nerka gill infected with Loma salmonae. Parasite DNA amplified with primers LS-1 and LS-2 (1% agarose gel, ethidium-bromide stained). The negative and positive controls (Ctr), respectively, used distilled water and DNA from purified L. salmonae spores. Fish were collected at Great Central Lake, Vancouver Island, British Columbia, Canada as migrating smolts (>=2 y old). Note nonspecific bands in lanes 5, 12 & 21. Molecular weight markers (bp) are shown in lane 1 145  xiii  PREFACE  Chapter I has been previously published as: Shaw RW, Kent ML (1999) Fish microsporidia.In: The microsporidia and microsporidiosis. ASM Press, Washington, DC, p 418-446.1 wrote the majority of the document in preparation for his final thesis. Dr. M.L. Kent wrote the sections entitled treatments and Nucleospora.  These sections were  updated and edited by myself. Sections of chapter II have been previously published as: Shaw RW, Kent ML, Adamson ML (1998) Modes of transmission of Loma  salmonae  (Microsporidia). Diseases of Aquatic Organisms 33:151-156.1 designed the experiment and wrote the article during preparation for my final thesis. Sections of chapter III are in press as: Shaw RW, Kent ML., Adamson ML (1999) lodophor treatment is not completely efficacious in preventing Loma salmonae experimentally challenged chinook Oncorhynchus  (Microsporidia) transmission in  tshawytscha  (Walbaum) salmon.  Journal of Fish Diseases. I designed the experiment and wrote the communication during preparation for my final thesis. Sections of chapter IV have been previously published as: Shaw RW, Kent ML, Docker MF, Brown AMV, Devlin RH, Adamson ML (1997) A new species of Loma (Microsporea) in shiner perch  aggregata).  (Cymatogaster  Journal of Parasitology 83:296-301.1 discovered and described the parasite  except in aspects related to genetic sequencing. These authors are in agreement as to the contributions of each.  RossW. STiaw  P Dr. M.L. Kent  Dr. M.L. Adamson  Amanda l\^V. Brown  xiv  ACKNOWLEDGEMENTS  I would like to thank Dr. M.L. Adamson for his support, guidance, and suggestion to only drink milk in life. I would also like to thank Dr. M.L. Kent for his enthusiasm, and many suggestions on experimental protocol and design. The Biological Station, run by the Department of Fisheries and Oceans in Nanaimo, British Columbia, was a wonderful place to conduct my research. The station possesses excellent facilities and I would like to specifically thank S. Dawe, J. Richard, C. Whipps, J. Dawson-Coates, G. Traxler, G. Prosperi-porta, J. Bagshaw, C. Westby, M. Higgins, D. Whitaker, T. McDonald, and Sophie St-Hilaire. A commercial salmon producer generously supported my research by providing infected and naive fish, and allowing me to sample their sites. Several other companies also participated in the project by submitting samples of fish for testing. I am indebted to my parents, fiendish friends, and other graduate students of the Adamson lab. I would specifically like to thank both Shannon Bennett whom I asked endless trivial favors of, and Paulette Chauvet who inspires me in life. Finally I would like to thank my committee for their helpful comments and Natural Sciences and Engineering Research Council of Canada Grant No. 582073 for funding my research.  xv  CHAPTER I Overview Fish-Infecting Microsporidia INTRODUCTION About 100 species and 14 genera of phylum Microsporidia Balbiani, 1882 have been described from fishes, and several cause severe disease. Microsporidia of fishes are widely distributed by both host species and geographic location. Whereas most fish microsporidia are host specific, at least at the genus level, a few (e.g. Glugea  stephani  Hagenmuller, 1899) show broad host specificity (Canning & Lorn 1986). For comprehensive lists of microsporidian species and their fish hosts see Canning & Lorn (1986), Lorn & Dykova (1992), and Dykova (1995). Herein I present an overview of economic importance, immunology, and treatment, followed by a review of the most important genera. My review focuses on microsporidia causing significant pathology in cultured fishes. Features of pathology and transmission relevant to individual microsporidia species are included under the genera section. For a general review of microsporidia transmission see Cali & Takvorian (1999). The ability of some microsporidia to cause disease in fish is well known. Increased mortalities in wild fishes such as rainbow smelt Osmerus mordax Mitchill (Haley 1952; Nepszy et al. 1978), young gizzard shad Dorsoma cepedianum LeSueur (Putz 1969; Price 1982), and freshwater salmonids (Putz et al. 1965; Urawa 1989) have been attributed to microsporidia infections. In wild fishes, declines of entire commercial fisheries have been attributed to microsporidiosis (Mann 1954; Sindermann 1966; Ralphs & Matthews 1986). Pleistophora macrozoarcides Nigrelli, 1946 has been implicated in the collapse of the North American ocean pout Macrozoarces  americanus  Schneider fishery (Fischthal 1944; Sandholzer et al. 1945; Sheehy et al. 1974). Declines in the rainbow smelt fishery of New Hamshire were due in part to infection by 1  G. hertwigi  Weissenberg, 1911 (in Haley 1954). Disfiguring of fish by large cysts or  liquefaction of muscle can decrease catch value (Nigrelli 1946; Grabda 1978; Egidius & Soleim 1986; Pulsford & Matthews 1991). Microsporidiosis can also cause indirect mortalities through starvation and reduced growth rates (Matthews & Matthews 1980; Figueras et al. 1992). Sprengel & Luchtenberg (1991) found that impairment of swimming ability in European smelt, O. eperlanus ladogensis  L , infected by  Pleistophora  Voronin, 1978 may contribute to higher mortalities due to predation. Wilkund  et al. (1996) reported that P. mirandellae  Vaney & Conte, 1901 which infects gonads,  reduced the reproductive capacity in roach Rutilus  rutilus  L. Selective mortalities in  juvenile stocks can further reduce reproductive capacity. For example,  Ralphs & Matthews, 1986 infects primarily the liver of juvenile grey mullet  hepaticus Chelon  Microgemma  labrosus  Risso, and may contribute to mortality (Ralphs & Matthews 1986).  An increase in aquaculture on a worldwide basis has led to the description of several new microsporidia and outbreaks of microsporidiosis in recent years. Compared to their wild counterparts, cultured fish are particularly susceptible to microsporidian infections because of high stocking densities. Extremely high mortalities have occurred in ornamental fishes as a result of infections by Glugea Pleistophora  hyphessobryconis  spp. (Lorn et al. 1995).  Shaperclaus, 1941 (Fig. 1) well known to fish hobbyists  as the source of neon tetra Paracheirodon  innesi  Myers disease, is one of the most  common parasites of aquarium fishes (Canning & Lorn 1986). Massive mortality of neon tetras held in aquariums is still not uncommon. Morphological abnormalities and mortality due to microsporidiosis in the ornamental zebra danio Brachydanio  rerio  have  also been reported (Kinkelin 1980). The zebra fish is now a very important laboratory animal for genetic research, and large colonies of this fish are being maintained at several universities in Europe and North America. ML Kent (pers. comm.) has observed 2  this microsporidian infection in zebra fish kept in European and North American colonies. The primary site of infection is the central nervous system, where it is often associated with chronic inflammatory changes (Fig. 2). In addition infections and associated severe inflammation in the skeletal muscle surrounding vertebrae and in the kidney have been seen (ML Kent pers. comm.). Heterosporis Pseudocrenilabrus  fish Betta splendens  multicolor  spp. can infect cichlid  Seegers (Lorn et al. 1989), ornamental Siamese fighting  Regan (Lorn et al. 1993), and angel fish Pterophyllum  Curs  scalare  & Valens (Michel et al. 1989) causing emaciation and distress or severe pathological changes in muscle tissue. Microsporidia are also important in cultured food fishes. Hatcheries raising salmonids for enhancement of wild stocks or for profit, have experienced epizootics of microsporidia (Putz 1969; Hauck 1984; Urawa & Awakura 1994). Mortality and disease have occurred at both freshwater (Markey et al. 1994) and marine sites where fish are grown to market size (Kent et al. 1989). Chronic low mortality in sea bream arurata  L., for example, has been caused by a Pleistophora  (Abela et al. 1996). Culture of Japanese eel Anguilla and ayu Plecoglossus Heterosporis  anguillarum  plecoglossiTakahashi  altivelis  japonica  Sparus  sp. that infects the muscle Temminck & Schlegel  Temminck & Schlegel is often plagued by infections of  (Hoshina, 1951) (syn. Pleistophora  anguillarum),  and  Glugea  & Egusa, 1977 (Awakura 1974; Kano & Fukui 1982; Kim et al.  1996) respectively. Production of minnows used for bait fishes is often affected by infections of P. ovariae  Summerfelt, 1964, which damage the ovaries of fish and thereby  reduce fecundity (Nagel & Hoffman 1977). Early investigations of fish microsporidia included observations of host response (Drew 1910; Debaisieux 1920; Nigrelli 1946). Researchers during that time period occasionally confused host reparative events (i.e. phagacytosis) with early parasite 3  development stages (Canning & Lorn 1986). Presently, host-parasite relationships of fish microsporidia can be broadly classified into two groups: those associated with xenoma formation (e.g., Glugea, Ichthyosporidium, Jirovecia, Loma, Microfilum, Microgemma,  Nosemoides, Spraguea, Tetramicra) (Fig. 3), and those associated with  non-xenoma forming microsporidia (e.g., Nucleospora, Heterosporis,  Pleistophora,  Thelohania) (Fig. 4). The term "xenoma" was developed from early works of Chatton (1920) and Weissenberg (1922). Cells infected by certain species of microsporidia become transformed and hypertrophied, resulting in a unique host cell-parasite complex. The host cell alters its structure and size, becoming physiologically integrated with the parasite. Cytoplasmic contents are replaced by the parasite, and the surface of the cell, or xenoma, becomes modified for increased absorption. Microvillus-like structures form in cells infected by Microsporidium cotti Chatton & Courier, 1923, Ichthyosporidium, and Tetramicra, while Glugea and Loma spp. utilize numerous pinocytotic vesicles (Canning & Lorn 1986). Hypertrophied cells can reach sizes of 400500 pm or larger, and are visible macroscopically as white cysts (Matthews & Matthews 1980; Ralphs & Matthews 1986) (Fig. 5). Eventually the cell is totally destroyed by the parasite. Spores released when a xenoma ruptures are easily identified by a large posterior vacuole in wet mounts and histological sections (Fig. 6).  IMMUNOLOGY The specific and nonspecific defense systems of fish are well reviewed by Kaattari & Piganelli (1996), Secombes (1996), and Dalmo et al. (1997). In comparison to the situation in viral and bacterial pathogens, the response of fish to microsporidian infections is relatively unexplored. Phagocytosis of spores by macrophages has been described for several 4  microsporidian species (Dykova & Lorn 1980; Canning & Lorn, 1986; Pulsford & Matthews 1991; Kim et al. 1996). Macrophages actively ingest and breakdown spores (Fig. 7), playing a crucial role in the host defense mechanism. The spore coat contains an outer protein layer and an inner chitin layer. Spores appear to be digested from the inside out indicating fish macrophages possess a wide repository of chitinases (Dykova & Lorn 1980; Canning & Lorn 1986). Microsporidia are not without their own defenses, as spores may prevent phagosome-lysosome fusion. Weidner & Sibley (1985) found that anionic components on the spores of G. hertwigi  may form ionic bonds with the  phagosome membrane, increasing rigidity and preventing fusion with a lysosome. It is not surprising that some microsporidia have evolved this protective mechanism as has occurred with other intracellular parasites, such as Chlamydia Brownridge 1978), and Toxoplasma  gondii  psittaci  (Wyrick &  Nicolle & Manceaux, 1908 (Jones & Hirsch  1972). Suppression of the host inflammatory response by microsporidia has also been documented (Dykova & Lorn 1978, 1980). Laudan et al. (1986a,b, 1987, 1989) found that spores of G. stephani,  when phagocytized, suppressed immunoglobulin (Ig) levels  of winter flounder Pseudopleuronectes  americanus  Walbaum by stimulating  macrophages to release prostaglandin and/or leukotrienes. This finding was substantiated by injecting serum from infected fish into healthy fish, which resulted in immunosuppression of healthy fish. Administering idomethacin after spore exposure, also failed to produce immunosuppression in flounder. Glugea  stephani  impairs the  host's humoral response to other infectious agents, and interferes with initiation, as well as with other levels, of the response. After G. stephanibecomes  established, the host's  immune system can recover. It has been suggested that Tetramicra  brevifilum  Matthews & Matthews, 1980 5  can immunosuppress turbot Scophthalmus that found in G. stephani  maximus  L. using a similar mechanism to  (Leiro et al. 1994). Figueras et al. (1992) found that the serum  agglutinin titers of naturally infected fish were low, although this did not increase their susceptibility to vibriosis {Vibrio  anguillarum).  These authors also isolated a serum  factor from infected fish, concluding that further study was necessary to determine if the factor was an immunomodulator or only a by-product of the parasite. Leiro et al. (1993) injected fish IP with whole spores or a crude extract of T. brevifilum.  At 30 d after  injection they gave the fish a second immunization of whole spores. They found that whole spores lead to higher antibody production initially, but that after 30 d fish responded more to crude extract antigens. Low seropositives were obtained from enzyme-linked immunosorbent assay studies on infected fish, leading them to suggest that this was indirect evidence of immunosuppression. Humoral and/or cellular response of a fish may also be impaired by infection with Nucleospora  salmonis  Heterosporis  anguillarum  Hedrick, Graff & Baxa, 1991 (Wongtavatchai et al. 1995b), or (Hung et al. 1997). Serum factors affecting T and B cells  directly may be secreted by these parasites, or parasites may stimulate release of mediators from macrophages. Leiro et al. (1996a) found that Glugea  caulleryiVan  den  Berghe, 1940 showed considerable homology of spore surface antigens to those of T. brevifilum.  However, G. caulleryidid  not suppress the humoral immune response of  turbot, but rather stimulated it during first and subsequent exposures. In general, caution should be exercised in assuming that lyphopenia (low lymphocyte numbers) is caused directly by a concurrent microsporidium infection because this condition may be caused by a wide variety of stressors (Barton & Iwama 1991; Figueras et al. 1992). The health of fish has often been used as a biological indicator of environmental conditions (Kent & Fournie 1993). Parasitic burdens can have significant impact on 6  mortality in wild fishes, and in some cases may be related to environmental conditions (Overstreet 1993). There have been very few studies dealing with the effects of anthropogenic contamination on microsporidian infections in fish. For example, Barker et al. (1994) found that abundance of G. stephani\n  winter flounder was significantly  higher at a pulp mill effluent site where pollutants accumulated in the sediment. In fish from polluted waters compared to fish from unpolluted waters, Barker et al. (1994) reported larger and more varied cysts forming in visceral organs, in addition to the intestine, which is considered the normal site of infection (Takvorian & Cali 1981). Chronic exposure to pulpmill effluent may have further immunosuppressed the flounder allowing G. stephani  to proliferate.  Immune responses by fish are often weak or nonexistent when microsporidia evade detection (e.g. by forming xenomas or simply by being intracellular). However, during initial infection, and especially during rupture of xenomas (see section on pathology) or pseudocysts (i.e. Pleistophora-type  Glugea  infections), microsporidia are  vulnerable to attack by the immune system of fish. Although antibody production in response to infection does occur (Buchmann et al. 1992; Leiro et al. 1993; Hung et al. 1996, 1997), it may not play a protective role (Kim et al. 1996,1997). Resistance to reinfection in recovered fish occurs in Loma salmonae Plistophora  salmonae  Morrison & Sprague, 1981 (syn.  Putz, Hoffman & Dunbar, 1965) (Speare et al. 1998b).  Different strains of the same fish species can exhibit varying degrees of susceptibility. For example, I have observed marked differences in susceptibility to L. salmonae  between different strains of chinook salmon Oncorhynchus  tshawytscha  Walbaum (Shaw et al. 1996). Phagocytes may play a primary role in preventing initial and subsequent infection by microsporidia. General phagocytic activity, measured by percent phagocytosis (PP, i.e. the percent of cells showing phagocytic activity or 7  percent of target particles engulfed), and phagocytic index (PI, i.e. the mean number of objects ingested per phagocyte), usually increases in the presence of serum. It is well known that opsonization by antibody and/or complement of nonself organisms enhances phagocytosis in fish (Griffin 1983; Sakai 1984; Scott et al. 1985; Olivier et al. 1986; Waterstrat et al. 1991; Matsuyama et al. 1992; Rose & Levine 1992; Pedrera et al. 1993; Leiro et al. 1996b). However, Leiro et al. (1996b) found that serum did not increase phagocytosis of G. caulleryior  T. brevifilum  by turbot macrophages. Neither  did macrophages from immunized turbot ingest significantly more spores than those from nonimmunized fish. They investigated this further, by treating spores with sodium m-periodate to block the binding of macrophages to surface-borne sugars on spores. These spores were ingested less effectively, suggesting that macrophages recognize sugars on the surface of the microsporidia. Lectinophagocytosis, the binding of sugars on the pathogen by macrophage receptors, has also been shown in tilapia spilurus  Oreochromis  Gunther by Saggers & Gould (1989). Leiro et al. (1996b) concluded that  microsporidia may modify the phagocytic response in fish. I have found that PP and PI of L. salmonae  spores by macrophages differ significantly between Atlantic salmon  Salmo  (which is completely resistant to L.salmonae)  salarL.  and the very susceptible  chinook salmon (see Chapter V). Temperature can also affect the ability of a fish host to mount an immune response, and phagocytic activity of macrophages is dependent on temperature (Finn & Nielson 1971; Leiro et al. 1995). Development in fish-infecting microsporidia can also depend on ambient temperature (Awakura 1974; Olson 1981; Speare et al. 1998a). For example, Speare et al. (1998a) were unable to detect L. salmonae trout Oncorhynchus  mykiss  infections in rainbow  Walbaum held at 10°C, although fish held at 14.5°C  developed infections. 8  TREATMENTS Several drugs have been used to treat microsporidian infections in fish, mostly on an experimental basis. Fumagillin, an antimicrobial agent developed for treating the microsporidian Nosema apis Zander, 1902 infections in honey bees, is the drug most widely used to treat microsporidiosis in fishes. The drug apparently acts by inhibiting RNA synthesis (Jaronski 1972). Kano et al. (1982) reported that fumagillin was effective against the microsporidian H. anguillarum in eels Anguilla japonica. The drug has been used also against N. salmonis infections in chinook salmon (Hedrick et al. 1991b) and Loma salmonae infections in chinook salmon (Kent & Dawe 1994). Fumagillin has also been used successfully to control several myxosporean diseases in fish (e.g., whirling disease, proliferative kidney disease, and sphaerosporosis) (Molnar et al. 1987; Hedrick et al. 1988; Szekely et al. 1988; Lauren et al. 1989; Wishkovsky et al. 1990; Yokoyama et al. 1990; El-Matbouli & Hoffmann 1991; Sitja-Bobadilla & Alvarez-Pellitero 1992; Higgins & Kent 1996). Various concentrations of the drug were employed in these studies, and on the basis of these reports, 3 to 10 mg fumagillin kg" of fish day" is the recommended dose for 1  1  salmonids. Higher concentrations or prolonged treatment (e.g., 30 to 60 d) may cause anorexia, poor growth, anemia, renal tubule degeneration and atrophy of hematopoietic tissues in salmonids (Lauren et al. 1989; Wishkovsky et al. 1990). Lower doses may be efficacious for some microsporidian infections in fish (e.g., Hedrick et al. (1991b)) controlled N. salmonis infections with oral fumagillin treatment at 1 mg of drug kg" of fish 1  day" for 4 wk). Kent & Dawe (1994) reported positive results for controlling L. salmonae 1  infections at 10 mg of drug kg" fish day" with a 4 wk treatment. Recent experiments with 1  1  the drug demonstrated that the infections can be controlled with lower doses of 2 or 4 mg of drug kg" of fish day" (Higgins et al. 1998). 1  1  9  Fumagillin is not heat-stable. Therefore, it is recommended that feed be coated with the drug, instead of incorporating it into feed during milling. The drug is not very soluble in water, but is very soluble in alcohol. In most studies, fumagillin was mixed with alcohol and sprayed on the feed; then the feed was coated with oil. An analog of fumagillin, TNP-470 (Takeda Chemical Industries, Ltd.), is a potent antiangiogenesis agent (Kusaka et al. 1994). Fumagillin and its derivatives appear to work by binding and inhibition of methionine aminopeptidase type II (Liu et al. 1998) which is the target for angiogenesis inhibitors AGM-1470 and ovalicin (Sin et al. 1997). Furthermore, in laboratory studiesTNP-470 has been shown to be effective against the mammalian microsporidia pathogens Encephalitozoon Schuitema, Didier & Terpstra, 1995, Vittaforma Nosema  corneum  Hartskeerl, Gool,  intestinalis  corneae  Silveira & Canning, 1995 (syn.  Shadduck, Meccoli, Davis & Font, 1990), E. cuniculiLevaditi,  & Schoen, 1923 and E. hellem  Nicolau,  Didier, Didier, Friedberg, Stenson, Orenstein, Yee, Tio,  Davis, Vossbrinck, Millichamp & Shadduck, 1991. Higgins et al. (1998) tested the efficacy of this drug in controlling L. salmonae  and N. salmonis  in experimentally  infected salmon. Fish treated orally at 1.0 mg or 0.1 mg of drug kg" of fish day" for 6 wk 1  1  showed markedly reduced infections by both microsporidia, even at the low dose. No significant toxic side effects were associated with the treatment. Higgins & Kent (1996, 1998) also found that treating fish at this low dose prevented infections in fish naturally exposed to the PKX myxosporean. The effects of a systemic triazinone, Toltrazuril (Bayer AG) on Glugea Moniez, 1887 infections in sticklebacks Gasterosteus  aculeatus  anomala  L. have been investigated  at the ultrastructure level (Schmahl & Mehlhorn 1989; Schmahl et al. 1990). Bath exposure of the drug was found to cause destruction of all life stages of the parasite, but the overall effects of this drug in reducing the prevalence or intensity of microsporidian infection in fish 10  have not been reported. Awakura & Kurahashi (1967) reported that amprolium inhibited merogony in Microsporidium takedai administered at 0.06 % body weight day" for up to 48 d, but the 1  treatment was associated with toxic side effects. There are a few other reports of treatment of fish microsporidia. For example, Andodi & Frank (1969) (cited in Canning & Lorn 1986) claimed that they cured P. hyphessobryconis  infections in aquarium fish by  adjusting the pH to 7.5-8.0 and introducing ozone at a rate of 1 mg h" 100 L" . Nagel & 1  1  Summerfelt (1977) reported that nitrofurazone reduced infections of Pleistophora ovariae in golden shiners Notemigonus chrysoleucas Mitchill when used in oral treatments at 2.2 - 3.3 g of drug kg" feed. Speare et al. (1998c) reported delayed L. 1  salmonae xenoma formation in rainbow trout fed to satiation with quinine hydrochloride treated feed (5 g kg" ). 1  Elevated water temperatures have also been used to treat some fish-infecting microsporidia. Takahashi & Ogawa (1997) reported an effective treatment for G. plecoglossi\n  ayu was to keep fish at 29°C for 5 d 10 to 20 d after initial infection and  repeat this treatment a week later. Kim et al. (1997) noted H. anguillarum in Japanese eel could also be treated with elevated water temperatures resulting in morphologically abnormal spores in the musculature. Normal spores were also recovered from treated fish suggesting relapses of infection might occur.  SIGNIFICANT MICROSPORIDIAN DISEASES Xenoma-Forming Genera G LUGE A Species of Glugea are some of the most intensively studied fish microsporidia. They infect the submucosal intestinal cells of a variety of wild and cultured fishes. 11  Disease in the food fish sand smelt Atherina  boyeri  Cuvier, and bait fish D. cepedianum,  lucioperca  Berrebi, 1978, G. luciopercae  Risso, pike perch  Stizostedion  can be caused by Glugea  atherinae  Dogiel & Bykhowsky, 1939, and G. cepedianae  Putz,  Hoffman & Dunbar, 1965 respectively (Price 1982; Canning & Lorn, 1986). However, most research has focused on G. anomala  in stickleback (Gasterosteus  spp.), G. hetwigiin  ayu, and G. stephaniln  (Pleuronectes  smelt, G. plecoglossi\n  spp. and Platessa  spp.). Glugea  anomala  and  Pungitius  flatfish  represents classical  developmental and pathological features (Weissenberg 1968; Dykova & Lorn 1980), while mortality of fishes have been documented in the other species (Olson 1976; Nepszy et al. 1978; Cali et al. 1986; Kim et al. 1996). Glugea  and G. plecoglossi  anomala  develop in almost all body organs of the  host, forming large (2 to 3 mm) white xenomas, which can cause serious injury to organs (Fig. 8). Glugea  hertwigi  (Fig. 9) and G. stephani  (Figs. 10 & 11) primarily infect  the subepithelial connective tissue of the intestine, although in heavy infections, G. hertwigi  can spread to all other organs, even the skeletal muscle and gills (Canning &  Lorn 1986).  Pathology.  Glugea  spp., like Loma, and Tetramicra,  form xenomas that can be  classified by stages of development (Dykova & Lorn 1980). Early or young xenomas have a cytoplasm filled uniformly with developmental stages of the parasite (Fig. 12). As they grow, the developmental stages occupy the center of the xenoma surrounded by peripheral host cytoplasm. For Glugea,  maturing xenomas exhibit a light retractile wall,  and peripheral host cell cytoplasm with spores in the center. A fully developed xenoma is filled with spores and few host cell components (Fig. 13). The host's reaction to xenoma formation varies depending on the stage of xenoma development, the tissue infected, and the age and species of host. However, some generalizations can be 12  made. The stages of host reaction, described below, were originally developed by Dykova & Lorn (1978) for G. anomala,  G. aculeatus,  G. hertgiwi,  G. plecoglossi,  and G.  Further study and clarification was made by Dykova & Lorn (1980), and  stephani.  Canning & Lorn (1986). Weakly  Reactive  Stage.  Early or very young xenomas begin to exert pressure atrophy on the surrounding tissue as they grow. The host responds by local proliferation of connective tissue and collagen fibers, forming a concentric layer of connective tissue around the xenoma. The weakly reactive stage is also seen with fully developed xenomas. Productive  Stage.  Mature xenomas elicit prominent inflammation (Fig. 14). Inflammation includes influx of fibroblasts and macrophages. Proliferative inflammation accompanies changes in the xenoma wall, although it is not clear if this is a causal relationship (Dykova et al. 1980; Canning & Lorn 1986). The wall of the xenoma swells, fibroblasts appear within it, and the wall ruptures and begins to disappear. In addition to fibroblasts, the formation of a capsule composed of eosinophilic granular cells around the xenoma was reported by Reimschuessel et al. (1987). Ichthyosporidium  spp. elicit a slightly different  inflammatory response characterized by epithelioid cells and histiocytes. Epithelioid cells encasing the xenoma are arranged with their long axis perpendicular to the xenoma wall. Destruction or rupture of the xenoma wall is accompanied by dystrophic changes that occur with the appearance of periodic acid-Schiff positive substances and Ca  2 +  within the mass of spores. The xenoma is then replaced by granulomatous tissue  (Fig. 15). Granuloma  Involution  Stage.  After the xenoma ruptures, spores are released and ingested by macrophages. 13  The granulation tissue matures and the granuloma gradually recedes. Fibrous connective tissue that has developed during the productive stage undergoes hyalinization, and the lesion is slowly resolved. Although the xenoma can be completely eliminated, functional restitution of heavily infected organs is not possible (Dykova et al. 1980). Glugea  spp. can cripple, disfigure, debilitate, and kill fishes (Sindermann, 1990;  Dykova 1995). These microsporidia can also contribute to decreased fecundity and retarded growth (Chen & Power 1972; McVicar 1975). The large cysts of G.  anomala  create serious tissue injury due to pressure atrophy, although sticklebacks appear to survive even heavy infections (Canning & Lorn 1986). Host response follows the stages described in this section under "pathology". Glugea  plecoglossi  has low organ  specificity, although development and host tissue reactions are similar to those of G. anomala  (Dykova et al. 1980). The prevalence of G. hertwigiln  smelt can reach 100% in  some lakes, and epizootics with intensities of 250 xenomas fish" have occurred 1  (Anenkova-Khlopina, 1920; Haley 1952, 1953, 1954; Bogdanova 1957; Petrushevski & Shulman 1958; Delisle 1965; Nepszy & Dechtiar 1972; Canning & Lorn 1986). Delisle (1972) estimated that in Lake Erie alone, 10 million fish y" were lost due to G. 1  hertwigi  infections. The intestinal epithelium can disintegrate, resulting in general septicaemia, intoxication, and death (Canning & Lorn 1986). The fecundity of smelt can also be severely affected by the parasite (Sindermann 1963). Infections by G. stephani  occlude  the intestinal lumen, disrupt its integrity, and contribute to emaciation or even death of the host (Cali et al. 1986). Xenomas replace the intestinal wall, forming a rigid layer up to 4 mm thick which has a chalk-white pebbled appearance (Fig. 11). Fish can die, even from low level infections, if xenomas form in the mucosa and rupture into the epithelial lining. In contrast, Cali et al. (1986) also showed that heavily infected fish may survive if 14  xenomas form in the serosal side of the intestinal tract. Olson (1976) reported collecting emaciated starry flounder Platichthys infected with G. stephani.  stellatus  Pallas from Yaquina Bay, Oregon,  Cali et al. (1986) also observed G. stephani\r\  moribund  winter flounder of Sandy Hook Bay, New Jersey. Heavy infections may be fatal in plaice Pleuronectes  L. held in aquariums or at fish farms (Buckmann 1952; McVicar  platessa  1975). McVicar (1975) suggested G. stephani  Transmission.  Glugea  may be highly contagious at a farm site.  spp., like most fish microsporidia, are transmitted directly via  ingestion. These spp. have been transmitted by intraperitoneal injection (IP) or via a crustacean paratenic host (e.g. transport hosts such as Daphnia  spp., brine shrimp,  amphipods) (Weissenberg 1921, 1968; McVicar 1975; Olson 1976; Kim et al. 1996). Predation may disperse spores of G. hertwigi,  or cannibalism may lead to infections in  other smelt (Haley 1954; Delisle 1972). Olson (1976, 1981) found that spores of G. stephani  pass directly through the crustacean digestive tract, and that infections heavier  than IP injection result when a crustacean paratenic host was used. He suggested that amphipods may represent a natural route of transmission for G. The target host cell of Glugea  spp. can vary. Glugea  stephani.  anomala  may target a  migratory mesenchyme cell, such as a macrophage or histocyte (Weissenberg 1968), whereas G. stephani  may infect neutrophils (Canning & Lorn 1986). The formation of  xenomas free within the body cavity by IP injection suggested to McVicar (1975) that G.  stephaniinfected  macrophages.  Seasonal and geographic variations in prevalence of G. stephanih&ve  been  related to water temperature (Olson 1976, 1981; Takvorian & Cali 1981, 1984). Olson (1981) found that development of the parasite was arrested at 10°C and development resumed when water temperatures were raised to 15°C or higher.  15  LOMA is another important xenoma-forming genus of microsporidia. Xenomas of  Loma Loma  spp., unlike those of Glugea,  develop asynchronously with various developmental  stages located throughout the xenoma. There are 9 described species of Loma, and this genus includes some species noted for infecting fishes of commercial importance (i.e., cod and salmon). Loma Nemeczek, 1911, Glugea  Nemeczek, 1911 (syn. Nosema  branchialis branchialis  branchialis  Nemeczek, 1911, and L. morhua  Morrison &  Sprague, 1981) have been described from several members of the family Gadidae: Gadus  aeglefinus  G. morhua  L , G. callarias  L , G. morhua  L , haddock Melanogrammus  marisalbiL.,  aeglefinus  G. morhua  L , and rockling  kildinensis  L,  Enchelyopus  L. (Bazikalova 1932; Dogiel 1936; Fantham et al. 1941; Shulman & Shulman-  cimbrius  Al-bova 1953; Morrison & Sprague 1981a,b; Morrison & Marryatt 1986). Loma camerounensis  Fomena, Coste & Bouix, 1992 infects tilapia Oreochromis  niloticus  L  which are widely cultured in West Africa (Fomena et al. 1992). Morrison & Sprague (1981c; 1983) described Loma Salvelinus  fontinalus  fontinalis  Morrison & Sprague, 1983 from brook trout  Mitchill in Canada. Loma salmonae  pathogen in farmed salmonids (Oncorhynchus  is well known as a serious  spp.) in the Pacific Northwest (Kent et al.  1989; Kent 1992; Shaw et al. 1997). This pathogen may have been introduced to Europe with rainbow trout and coho Oncorhynchus  kisutch  Walbaum transported from  Californian stocks (Poynton 1986). Descriptions of Loma spp. from salmonids such as chinook, coho, and rainbow trout (Awakura et al. 1982; Hauck 1984, Mora 1988; Speare et al. 1989; Magor 1987; Gandhi et al. 1995) are likely L. salmonae  based on host,  parasite morphology, and site of infection. All of these species except L. camerounensis  infect endothelial cells causing  formation of xenomas throughout vascularized organs (eg. heart, kidney, spleen, liver) 16  but primarily in the gills (Fig. 5). Loma  camerouensis,  forms xenomas in connective  tissue of the gut submucosa. Species descriptions for Loma have been problematic. Most earlier researchers designated new parasite species based on a new host, geographic location, and slight differences in spore morphology. Morrison & Sprague (1981a,b,c; 1983) described L. and L. fontinalis  morhua  and L. salmonae,  branchtalis  of Loma  recognizing that these species may be conspecific to L. respectively (Canning & Lorn 1986). Analysis of the rDNA  spp. and transmission studies, are beginning to clarify the taxonomy of this  genus (Docker et al. 1997a; Shaw et al. 1997). Shaw et al. (1997) described L. embiotocia  Shaw, Kent, Docker, Brown, Devlin and Adamson, 1997 in shiner perch  Cymatogaster  aggregata  Gibbons with all these techniques. They found that the internal  transcribed spacer (ITS) region of L. embiotocia region of L. salmonae  differed significantly from the ITS  but not within isolates of each species. Furthermore, they were  unable to transmit L. salmonae  to shiner perch and thus assigned the shiner perch  parasite as a new species, L. embiotocia.  I have been unable to transmit L.  to chinook, or Loma spp. isolated from Pacific cod Gadus ling cod Ophiodon  elongatus  macrocephalus  embiotocia  Tilesius, or  Girard to chinook. This suggests that Loma spp. being  found in marine hosts (Kent et al. 1998) may be distinct species, or are all at least distinct from L.  salmonae.  Wales & Wolf (1955) found 75% of wild yearling rainbow trout were infected with L. salmonae  in California. I have observed L. salmonae to be widespread in wild Pacific  salmon populations in both fresh and marine habitats in British Columbia, Canada (see Chapter VI). Epizootics of L. salmonae  have been recorded in Japan, the Pacific  Northwest, the eastern United States, and Europe (Wales & Wolf 1955; Hauck 1984; Bekhti & Bouix 1985; Canning & Lorn 1986; Kent et al. 1989; Bruno et al. 1995). Wales 17  & Wolf (1955) first described transport and grow-out of farmed fish being more difficult because of L. salmonae.  Since that time the parasite has continued to cause large  economic losses to chinook farms in British Columbia. Recently, high mortality in wild spawning sockeye of the Babine Lake system, British Columbia occurred in association with heavy L. salmonae  infections (Higgins MJ pers. comm.).  I have found that L. salmonae  infects all 7 species of Oncorhynchus,  and brook  trout (see Chapter IV). Speare et al. (1998a) were unable to infect brook trout, although their fish may have developed resistance from a previous exposure to L. salmonae (DJ Speare pers. comm.). Atlantic salmon, Arctic char Salvelinus  fontinalis  herring Clupea  pallasi  Valenciennes, prickly sculpin Cottus  spined sticklebacks, and guppy Poecilia by L. salmonae  reticulata  asper  host specificity with the main hosts being species of the genus branchialis  L.  Richardson, three-  are resistant to experimental infection  (Kent et al. 1995a; Chapter IV). Loma salmonae,  Pathology. Cod infected with L.  alpinus  or L.  therefore, displays Oncorhynchus.  exhibit no obvious clinical signs (Morrison  & Sprague 1981a). However, xenomas in the gills reach 1.2 mm in size, resulting in distortion and displacement of gill tissue and blood vessels (Kabata 1959; Morrison & Sprague 1981a). Pathological changes caused by these infections are similar to those described for G. anomala,  except that Morrison (1983) found that spores remained in  the center of granulomas with both phagocytes and fibroblasts undergoing coagulative necrosis. Impact on host mortality is unknown, although infections may affect metabolism negatively through decreased respiratory efficiency (Morrison & Sprague 1981a). Gross clinical signs of L. salmonae  infection may include: darkening of the tail or  body, lethargy, gill pallor, petechial haemorrhaging in the gills, skin and fins, ascites, hemorrhagic pyloric caeca; and white cysts (xenomas) on the gills (Hauck 1984; Kent 18  1992; Markey et al. 1994; Bruno et al. 1995). Reduced growth, impaired swimming efficiency, and increased mortality of young chinook have also been noted (Hauck 1984). The growth rate in rainbow trout appears to be reduced during xenoma formation (DJ Speare pers. comm.). Hauck (1984) provides a comprehensive pathological description of a Loma salmonae  systemic infection, that includes necrosis of cartilage  and musculature, occlusion of arteries, pericarditis of the bulbus arteriosus, and hyperplasia of gill and heart tissues. The effects of occlusion are seen when xenomas extend through the tunicae media and adventitia of arteries. Xenomas and/or free spores in the heart can result in mural emboli, hyperplasia of ventricular and atrial tissue, and an oligocythaemic condition. Gill xenomas can cause subacute to chronic vasculitis, perivasculitis, vascular thrombosis, and hyperplasia of gill tissue (Kent et al. 1989; Speare et al. 1989). As with other xenoma-forming species, the inflammatory response and associated tissue damage is more severe after xenomas have ruptured (Kent et al. 1989). Fomena et al. (1992) did not describe pathology of L. camerounensis  except to  note a high prevalence (94%) of the parasite and that large xenomas could protrude into the intestinal cavity. Negative impacts on fish health were also not described by Morrison & Sprague (1981c, 1983) for L. fontinalis. similar to that of L.  It is likely that the pathology is  salmonae.  Transmission. Spores of  Loma  spp. released from xenomas can be infectious when  ingested. Spores may be liberated directly from the gills and urine into the external environment (Hauck 1984). Autoinfection may occur by spores released from ruptured xenomas being transported to other tissues. Morrison (1983) proposed this phenomenon for L. branchialis.  Shaw et al. (1998) substantiated this for L. salmonae  by  experimentally inducing infection by injecting purified spores intravascularly. Hauck 19  (1984) suggested that infection may occur by direct phagocytic uptake of spores by the gills. However, placement of infectious spores directly on gills of chinook did not result in infection (Shaw et al. 1998). Salmon may also be infected experimentally by anal gavage, peros,  intravascular (IV), intramuscular (IM), and IP injection (see Chapter II).  Poynton (1986) and Kent et al. (1995a) suggested that L. salmonae  is transmitted from  fish to fish within a freshwater or marine netpen. Fish have been infected by cohabitation with carrier fish in a flow through system, suggesting that direct fish-to-fish transmission occurs within a net pen (Shaw et al. 1998). The infection is prevalent in wild ocean-caught salmonids (Kent et al. 1998), which may be a source of infection for marine fish farms. However, infections are probably maintained and perpetuated at these farms by stocking of parasite-free smolts at the same sites with older, infected fish. Chinook can remain infected for extended periods of time (i.e. > 80 d at 10°C) and purified spores of L. salmonae  are viable for 85 d in both fresh and sea water (see  Chapter III). Epizootics of L. salmonae  have occurred at freshwater hatcheries (Hauck  1984; Magor 1987) indicating smolts may also be infected before they are transferred to seawater farms. Development of immunity to L. salmonae  occurs in rainbow trout. Speare et al.  (1998b) found that fish resolved experimental infections by 10 wk after initial exposure and were resistant to experimental reinfection. My preliminary observations suggest that the same phenomenon can occur with chinook salmon. The structure and development of xenomas formed by Loma  spp. have been  studied extensively. However, very little is known about the route the parasite takes after entering the gut of the host (see Chapter II). It is likely that sporoplasms from extruded spores are injected into epithelial cells of the alimentary canal. Shaw et al. (1998) observed intracellular structures, possible sporogonic stages, progressing 20  though intestinal epithelial cells and into the lamina propria. Markey et al. (1994) described unidentified intracellular structures preceeding xenoma formation in all infected tissues. These structures may have represented earlier stages of the parasite. Work is currently underway using in-situ hybridization with the rDNA probe (Docker et al. 1997a) to clarify the route of early L. salmonae  infections (DJ Speare pers. comm.).  TETRAMICRA Tetramicra  brevifilum,  another xenoma forming microsporidian, has been noted  to cause significant disease in cultured and wild fish. The host fish, turbot, is becoming more widely cultured in countries such as Spain, and the parasite represents a significant threat to turbot culture (Figueras et al. 1992). Tetramicra  brevifilum  infects  connective tissue of the skeletal musculature (Fig. 16), and in heavy infections xenomas can be found in the intestine, kidney, liver and spleen (Estevez et al. 1992; Figueras et al. 1992). Matthews & Matthews (1980) first described this parasite in wild turbot collected off the north coast of Cornwall, United Kingdom. Figueras et al. (1992) next isolated the parasite in turbot cultured in Galicia, Spain during 1990. Recently Maillo et al. (1998) found T. brevifilum  in a new host, Lophius  budegassa  Spinola in the  Northwest Mediterranean sea. Xenomas lack the multilaminate layer characterizing Glugea  spp. However, xenomas of T. brevifilum  may reach 1.5 mm in diameter with  additional adhesions between xenomas creating composite cysts (Matthews & Matthews 1980; Dykova & Figueras 1994). Spores of this species contain a conspicuous inclusion in the sporoplasm and posterior vacuole, which is unique among fish-infecting microsporidia (Lorn & Dykova 1992).  Pathology. Turbot with heavy infections of  T. brevifilum  exhibit erratic swimming  behavior, general tissue swelling, dorsal darkening, overproduction of mucus, muscle 21  liquefaction, visible cysts, and chronic low mortality. A 50% reduction in growth rate and a jelly-like consistency of muscle in infected stocks contribute to decreased marketability (Figueras et al. 1992). The impairment of swimming ability could contribute to decreased feeding rates in cultured stocks and increased predation and starvation in wild stocks (Matthews & Matthews 1980). The presence of such large xenomas creates pressure atrophy consistent with trauma in the perimysium (connective tissue of muscle fibers), leading to displacement of muscle fibers and loss of fiber attachment to myocommata or myomeres (Matthews & Matthews 1980). Canalization in connective tissue of the spinal column, and localized haemorrhaging, fibrosis, and necrosis of infected tissues occur with this infection (Matthews & Matthews 1980; Estevez et al. 1992). Rupture of xenomas results in general cellular infiltration and collagen deposition, and spores are phagocytized by macrophages and subsequently destroyed. Myodegeneration by vacuolization of the sarcoplasm and separation of myofibrils can create liquefaction of musculature. As in Pleistophora-Xype infections, this damage is thought to be caused by substances secreted by the parasite (Dykova & Lorn 1980). Estevez et al. (1992) noted that degeneration of the muscularis mucosa occurred but to a lesser extent than in skeletal muscles. Fish that survive infection may recover fully (Estevez et al. 1992). Figueras et al. (1992) and Leiro et al. (1993) investigated the effects of T. brevifilum on the immune status of fish (see "Immunology" previously). Transmission. Matthews & Matthews (1980) were able to transmit T. brevifilum by IM injection, but not by per os. No cross transmission to controls was observed by these researchers during the 7 wk fish were held at 15°C. They suggested that phagocytes may be involved in the life cycle of T. brevifilum by acting as a transport mechanism within the host, which could transport the parasite through the endothelium, or act as 22  sites for development themselves. Phagocytes might become directly infected by a sporoplasm or during phagocytosis of meronts in the lamina propria of the intestine. Estevez et al. (1992) observed free spores within the lamina propria of the intestine. Figueras et al. (1992) were unable to infect fish by IP or by waterborne exposure. No microsporidia were found in the food fed to cultured turbot, leading them to conclude the fish were infected by aquatic crustaceans such as copepods, decapod larva, or mysids. In their study, infection corresponded with a drop in temperature, possibly reflecting stress induced in the fish. The death of the host and the release of spores directly into sea water and/or cannibalism of moribund fish may be important in transmission of T. brevifilum.  Frequent mortality in the juvenile population could ensure  transmission of the parasite to other fish (Matthews & Matthews 1980).  Non Xenoma-Forming Genera PLEISTOPHORA Pleistophora,  like Glugea, causes disease in a wide variety of fish species. These  parasites usually invade skeletal muscles, replacing the sarcoplasm and destroying the cells. In general, they are associated with musculature destruction or liquefaction, deformity, and production of tumour-like masses in fish (Pulsford & Matthews 1991). Some species, such as P. mirandellae  and P. ovariae, invade and destroy oocytes.  Pleistophora  spp. are characterised by diffuse infections, with only a few species such as P. senegalensis  Faye, Touguebaye & Bouix, 1990, and P. hippoglossoideos  Bosanquet,  1910 forming xenomas (Morrison et al. 1984; Faye et al. 1990). More than 30 species of  (  Pleistophora  have been described. Of these, 8 infect fishes of economic importance and  include: P. ehrenbaumi  Reichenow, 1929 in wolffish Anarhichas  spp.; P.  Leiro, Ortega, Iglesias, Estevez & Sanmartin, 1996 in blue whiting  finisterrensis  Micromesistius  23  poutassou  Risso; P. hippoglossoideos  P. hyphessobryconis mirandellae senegalensis  in plaice Hippoglossoides  in ornamental fishes; P. macrozoarcides  in European cyprinids; P. ovariae in sea bream. Heterosporis  discussed with other species of  Fabricius;  platessoides  in ocean pout; P.  in bait fishes (family Cyprinidae); and P.  anguillarum  (formerly P. anguillarum),  is  Heterosporis.  Muscle fibers of wolffish A. lupus and A. minor are infected by P. ehrenbaumi  and  up to 10% of wolffish in the waters of Iceland may be infected (Meyer 1952). The parasite creates tumour-like swellings up to 8 x 15 x 4 cm, which can make the commercial catches unfit for consumption (Egidius & Soleim 1986). Wolffish are a promising species for aquaculture (Wiseman & Brown 1996), and thus this parasite may ultimately become a problem in the rearing of these fish. Blue whiting collected off the coast of northwest Spain have been infected by P. finisterrensis.  Leiro et al. (1996c) found 5% of fish harbored infections in the hypoaxial  musculature, in which infective 3 to 6 mm foci were located. They did not associate any significant morbidity with the infection. The type host for P. hippoglossoideos Drepanopsetta  hippoglossoides)  is H. platessoides  (syn. H.  limandoides,  (Bosanquet 1910; Kabata 1959; Canning & Lorn 1986).  Canning & Lorn (1986) and Lorn & Dykova (1992) also included sole Solea solea Quensel as a host. Muscles of the fins, walls of the visceral cavity, and somatic musculature of fish may be infected (Fig. 4). Cystlike structures are visible externally and can be up to 2.5 x 10 mm, in some cases, making the fish unfit for eating (Canning & Lorn 1986; Dykova 1995). Morrison et al. (1984) provide a redescription of this species. Originally imported from wild fishes of the upper Amazon river, P. hyphessobryconis  is now distributed world wide in many families of freshwater tropical  fishes, infecting over 16 species (Lorn & Dykova 1992; Dykova 1995). It invades skeletal 24  muscle (Fig. 1) and heavy infections can include connective tissue of the ovaries, intestinal epithelium, skin, and renal tubules. Large cysts (2 mm) can form, and spores can concentrate in the subcutaneous tissue and skin (Canning & Lorn 1986). Heavy losses occur in culture ponds of ornamental species infected by P. hyphessobryconis,  with few  successful treatments available (see section 1.3). Skeletal muscles of ocean pout infected with P. macrozoarcides  can contain  tumourlike masses up to 8 cm in size that exude a pus-like fluid when cut (Canning & Lorn 1986). These pseudocysts increase with age and size of fish and have been blamed for the 1940's collapse of the ocean pout fishery (Fischthal 1944; Sandholzer et al. 1945) as a result of difficulties in marketing fish infected with P. macrozoarcides  to consumers  (Sheehy et al. 1974). Pleistophora  mirandellae  (syn. P. longifilisSchuberg,  1910,  P.  oolytica\Ne\ser,  1949) infects oocytes of common European cyprinids such as bleak Alburnus barbel Barbus  barbus  L., and roach Rutilus  in common pike Esoxlucius  rutilus  alburnus  L.,  (Dykova 1995). It has been found also  L (Maurand et al. 1988). Macroscopic white lesions appear in  the ovary, and an infection of 10 to 20% of follicles can significantly decrease fecundity of fish (Lorn & Dykova 1992). Wilkund et al. (1996) reported that roach in the Archipelago Sea (Finland) infected with P. mirandellae  likely experienced decreased fecundity as a  result of ovary degeneration and destruction. Golden shiner and fathead minnow Pimephales hosts of P. ovariae  promelas  Rafinesque are the main  (Fig. 17). Summerfelt & Warner (1970) found an overall prevalence of  48% in shiner from U.S. bait-minnow hatcheries they surveyed. The lowest prevalence was found in young fish, with infection rate increasing up to 79% for older fish. The prevalence then dropped off, possibly as a result of selective mortality. Although this parasite was widespread in the golden shiner, hatcheries were able to obtain eggs from 25  young fish not yet damaged by P. ovariae  (Nagel & Hoffman 1977). The parasite may  cause a 40% decrease in fecundity (Summerfelt 1964). Sea bream is an important species in markets of Senegal. Faye et al. (1990) described P. senegalensis  from the muscularis of this host's intestinal wall, but no  significant pathological changes were associated with infection.  Pathology. In non-xenoma-forming genera such as  Pleistophora,  infected cell contents  are replaced without inducing prominent cell hypertrophy. These microsporidia infect muscle cells or oocytes, and the host reaction is often minimal. Minor inflammatory cell infiltration into the myosepta can take place as the parasite develops. Fusion of infected muscle fibers can also occur with some parasite species (eg. Pleistophora and P. macrozoarcidis).  ehrenbaumi  Eventually all the muscle cell contents are replaced and the cell  destroyed, liberating mature spores. The host then responds with an influx of macrophages that phagocytize and digest the spores, but tissue regeneration is limited (Dykova & Lorn 1980; Pulsford & Matthews 1991). Invaded muscle fibers are often not isolated by granuloma formation, as is the case in the xenoma-forming species. However, granulomas can develop as a result of heavy infections by certain species (eg.  P. macrozoarcidis,  P. hyphessobryconis).  Pleistophora  hyphessobryconis  is unique  in that it forms discrete islets of degenerated sarcoplasm in the muscle that directly abuts intact myofibrils. These islets result from substances secreted by the parasite (Dykova & Lorn 1980).  '  Pathologic changes have not been described for P. ehrenbaumi  infections.  However, fish can become extremely emaciated, suggesting that such infections can be fatal (Egidius & Soleim 1986; Lorn & Dykova 1992). Mortality due to P.  hippoglossoideos  in smaller individuals has been implied, but not confirmed (Morrison et al. 1984). Large cysts of P. hippoglossoideos  can cause compression and distortion of tissues, but no great 26  damage results (Kabata 1959; Canning & Lorn 1986). Morrison et al. (1984) observed influx of phagocytes and engulfment of spores in infected muscle, and some nodules were also encapsulated with fibrous tissue. Fishes infected with P. hyphessobryconis  show anomalous behaviour and  movement, fading of color, and appearance of greyish-white patches in the muscle. Emaciation, scoliosis, kyphosis, and bristled scales are also characteristic of the infection (Canning & Lorn 1986). Granulomas can form in the mesenteries or viscera with an envelope of connective tissue cells. Canning & Lorn (1986) found connective tissue encapsulation was better at limiting spread of the parasite than phagocytosis by macrophages. Heavy infections can cause high mortality within 14 d (Thieme 1954, cited in Canning & Lorn 1986). Destruction of the host muscle begins with a unique halo formation around the parasite, which consists of disrupted smooth endoplasmic reticulum, free ribosomes, and myofibrils (Canning & Lorn 1986; Dykova 1995). Infections can lead to atrophy of testes (parasitic castration) and liver, although these organs do not have to be heavily infected (Lorn & Dykova 1992). Canning & Lorn (1986) describe the pathology of P. macrozoarcides  infections. The  parasite can form small, whitish cylinders in muscle fibers during early stages of infection, but large tumour-like masses result when the host encapsulates several infected fibers with concentrically arranged connective tissue. The center of these "pseudocysts" contains free spores. The muscle turns brownish, and muscle fibers are hyalinized and destroyed. The host response follows that described earlier for other non-xenoma forming species. Both P. mirandellae  and P. ovariae  infect oocytes of fishes, although the  pathogenesis of these infections differs. Pleistophora radiata  mirandellae  infects before the zona  forms, developing in the yolk and replacing it with spores. A significant proliferative  granulomatous inflammatory response can occur, with destruction of infected oocytes and 27  spores (Canning & Lorn 1986; Dykova 1995). Infected connective tissue of the seminiferous tubules can also create hypertrophy of local epithelial cells leading to decreased fecundity (Schuberg 1910). In contrast, inflammation and encapsulation of infected oocytes is not associated with P. ovariae  infection. The inflammatory reaction has  not been well described for this species, although Summerfelt & Warner (1970) provide a brief description of its pathogenesis. Infected ova become mottled with white spots and streaks, each representing a mass of ovarian stroma and spores. Atresia of heavily infected ova occurs, followed by spores coalescing in a stroma of zona radiata  and yolk.  Hyperplasia of the follicular epithelium occurs, and then a collapse of the zona radiata, and an influx of phagocytes that destroy the oocyte. Atretic follicles of the ovary are invaded by fibroblasts and some fibrosis can occur. Infected ova develop a stroma of connective tissue and increase in size, as reflected by postspawning fish having heavier ovaries compared to healthy fish. Parasitic castration can be pronounced, reducing fecundity by 37% or more (Canning & Lorn 1986). Curiously, infected fish are often larger than healthy spawning fish, because of reduced commitment of nutrients to egg production (Summerfelt & Warner 1970). Transmission. As with many microsporidia, death of the host may liberate free spores of Pleistophora  spp. to infect the next host. Spores may also be released with urine, or  directly from the skin, of fishes infected with P. hyphessobryconis Tetras and goldfish have been infected peros, P. hyphessobryconis  (Canning & Lorn 1986).  and IM injections are helpful in maintaining  in the laboratory (Lorn 1969; Canning & Lorn 1986). Autoinfection, in  which spores produced within the host hatch and form secondary infections, has been proposed for P. hyphessobryconis. the host by P. macrozoarcides  Nigrelli (1946) proposed that infection of large areas of could be explained by autoinfection. It is likely that  autoinfection occurs in some Pleistophora  spp., but this has yet to be confirmed. 28  Summerfelt (1972) infected golden shiners with P. ovariae peros,  and suggested  transovarial (vertical) transmission after finding the microsporidian in 5% of 38 blastulas he examined. Canning & Lorn (1986) did not consider this observation definitive evidence for transovarial transmission. Interestingly, Leiro et al (1994) infected turbot peros whiting. Outbreaks of P. finisterrensis  with P. finisterrensis  from blue  may occur in turbot culture in the future as raw blue  whiting is often used as feed.  HETEROSPORIS  There are 3 species infecting fishes: Heterosporis schuberti  finki Schubert, 1969, H.  Lorn, Dykova, Kbrting & Flinger, 1989, and H. anguillarum.  The first two infect  ornamental fishes, whereas the latter is an important pathogen in eel culture. Heterosporis  finki infects the musculature of angel fish Pterophyllum  (Schubert 1969; Michel et al. 1989), a popular aquarium fish. Heterosporis has been described from musculature of Pseudocrenilabrus and Ancistrus  cirrhosus  multicolor(family  scalare schuberti  Cichlidae)  Eigenmann (family Loricariidae) (Lorn et al. 1989). Both species  are characterized by formation of a sporophorocyst which is a thick, dense envelope of parasitic origin inside the sarcoplasm (Fig. 18) (Lorn et al. 1993). The sporophorocyst contains only parasite stages, unlike a xenoma, which contains host cell cytoplasm and nuclei. Heterosporis  anguillarum  development resembles that of Pleistophora  spp. (see  species under this genus). Pathology. Michel et al. (1989) noted that few fish exhibited clinical signs during an outbreak of H. finkiat an angel fish farm in France. However, some fish were emaciated, and exhibited lesions up to 5 mm diameter. Liquefaction of infected striated muscle was also seen, although internal organs appeared normal. Lorn et al. (1989) 29  described signs of distress, emaciation, and up to 95% mortality of fish infected with H. schuberti.  Sporophorocysts provoked only moderate cellular infiltration. Rupture of  these sporophorocysts however, resulted in the formation of aggregates of macrophages containing phagocytized spores in the mesenteries, intestine, and kidney. A more severe reaction in B. splendens  infected by a Heterosporis  sp. was described by  Lorn et al. (1993). In these fish granulomatous myositis was characterized by a central area of lesions containing spores. Recovery of muscle function was deemed impossible. The growth of Japanese eels can be retarded and market value decreased by H. infection. Eels mount an extensive inflammatory response when cysts  anguillarum  rupture followed by a fibroblastic response. Deformities and muscle liquefaction can result from such infections (T'sui & Wang 1988). Transmission. Little information is available on transmission of Heterosporis  spp.  Michel et al. (1989) hypothesized that clinically normal adult angel fish transferred H. finkiXo  juveniles or that the infection was introduced with a food source. Infection in eels  is possible by oral administration of H. anguillarum  spores (Kano & Fukui 1982), or by  water-borne exposure (T'sui et al. 1988).  MICROSPORIDIUM  The collective group Microsporidium  contains many species that infect fishes,  some of which may not even belong to the phylum Microsporidia. For example, Microsporidium  pseudotumefaciens  Pflugfelder, 1952, infects common freshwater  aquarium fishes in the genera Brachydanio, Molliensia,  and Xiphophorus.  Colisa,  Lebistes,  Gambusia,  Platypoecilus,  It was thought to cause considerable mortality but has  been reported only once (Pflugfelder 1952, cited in Canning & Lorn 1986). Canning & Lorn (1986) transferred this parasite to the group Microsporidium  but pointed out that its 30  life cycle did not fit that of a microsporidian. Both Beko disease in yellowtail Seriola caused by M. seriolae  takedaiAwakura,  quinqueradiata  Egusa, 1982 (syn. Pleistophora  Temminck & Schlegal  sp. Ghittino, 1974), and M.  1974 in freshwater salmonids infect muscle and can be pathogenic in  commercially important fishes (Awakura 1974; Egusa 1982). Microsporidium  takedai  has been recorded from various salmonid hosts and geographic locations in Japan (Takeda 1933; Awakura et al. 1966; Kubota 1967; Funahashi et al. 1973; Awakura 1974,1978; Vialova 1984). Hosts for M. takedai keta Walbaum, pink O. gorbuscha  include: chinook, chum  Oncorhynchus  Walbaum, kokanee O. nerka Walbaum, and masu O.  masou  Brevoort salmon, white-spotted char Salvelinus  Salmo  trutta  L , dolly varden S. malma  leuconaenis  Pallas, brown trout  Walbaum, and rainbow trout. Kokanee salmon,  and rainbow trout are particularly susceptible to infection (Urawa & Awakura 1994). Urawa (1989) found that hatchery-reared masu salmon smolt were very susceptible to M. takedai  infection when released into rivers. Susceptibility is age dependent, with  young fish being highly susceptible (Awakura 1974). The prevalence of M. takedai  \s  also seasonal, peaking during the months of highest water temperature. Urawa (1989) found prevalence ranged from 80 to 100% in wild and hatchery-reared salmon from 1982 to 1984. Pathology. Beko disease is characterized by cyst formation in the muscle. Small and large depressions on the lateral surface of yellowtail can indicate the presence of M. seriolae  cysts (Canning & Lorn 1986). Spores can be found within cysts bounded by a  fibrous host membrane (Egusa 1982). The parasite causes muscle liquefaction, which undoubtedly contributes to lower marketability of infected yellowtail. Microsporidium  takedai  also forms cysts, which are located in the heart during  chronic infections, and the trunk, fin, jaw, eye, throat, and gullet muscles during acute 31  infections (Figs. 19 & 20) (Awakura 1974). Cysts of M. takedaiare spindle shaped (2 to 3 mm wide, 3 to 6 mm long) and lack a xenoma wall (Awakura 1974; Urawa & Awakura 1994). Both acute and chronic infections, with associated mortality, have been recorded from wild and hatchery-reared fishes. Chronic infections are characterized by cyst formation causing extreme hyptertophy, multiplication of connective tissue, vacuolization, and deformation of the heart (Fig. 19) (Urawa 1989; Urawa & Awakura 1994). Acute infections include cyst formation in general skeletal musculature that can result in considerable necrosis. Acute infections have also been associated with extremely high mortality in hatchery fishes (Awakura 1965; Urawa & Awakura 1994). Urawa (1989) observed a strong correlation between the condition of fish and the intensity of M. takedai infections. Hosts mount a typical inflammatory response to the parasite, and formations of granulomas are observed (Awakura 1974). Transmission. Little information is available on transmission of M. seriolae. Awakura (1974) transmitted M. takedai by peros, and by water-borne infection and suggested that paratenic hosts such as rotifers EuchIan is spp., and glochidia larvae of mussels Margaritifera spp. may play a role in transmission. However, Canning & Lorn (1986) noted the organisms he described in potential paratenic hosts were not microsporidia. Temperature plays a significant role in transmission and development of M. takedai. Development may be arrested at 8°C and retarded between 11 and 15°C (Awakura 1974). A successful control strategy has been to rear smolts below 15°C (Urawa & Awakura 1994), and fish that have recovered from infection can develop immunity for up to 1 y (Awakura & Kurahashi 1967).  NUCLEOSPORA  Nucleospora salmonis is a remarkable microsporidium that causes disease in 32  salmonid fishes. It infects the nuclei of hemoblasts, particularly lymphoblasts or plasmablasts. Infected cells exhibit massive proliferation and are immature, suggestive of neoplasia. This microsporidium was first observed in pen-reared chinook in Washington State, where it was associated with anemia (Elston et al. 1987). The parasite has also been reported in freshwater-reared chinook, kokanee, and steelhead trout (Morrison et al. 1990; Hedrick et al. 1990; 1991b). The infection is common in caged-reared chinook salmon in British Columbia (Kent 1998), and in Atlantic salmon in Chile (Bravo 1996). Intranuclear microsporidia very similar to N. salmonis lumpfish Cyclopterus hippoglossus  lumpus  have been observed in Atlantic  L. (Mullins et al. 1994), and Atlantic halibut  Hippoglossus  L. (Nilsen et al. 1995), and these organisms were tentatively assigned to the Enterocytozoon.  genus  The intranuclear microsporidian of salmonids was originally described as N. salmonis  by Hedrick et al. (1991 a), but was described shortly thereafter as  salmonis  by Chilmonczyk et al. (1991). Ribosomal DNA sequence comparison of N.  salmonis  and E. bieneusi  Enterocytozoon  Desportes, Charpentier, Galian, Bernard, Cochand-Priolett,  Lavergne, Ravisse, Modigliani, 1985 does not provide compelling evidence to suppress the nominal genus Nucleospora.  Although intrageneric sequence comparisons among  microsporidia are limited, N. salmonis  and E. bieneusi  (at 20.1% genetic divergence in  the 16S and 28S genes) show greater differences than congeneric species examined to date (Docker et al. 1997b). Differences in host and site of infection also support the separation of Nucleospora  from Enterocytozoon.  whereas Enterocytozoon  Nucleospora  develops within the nuclei of fishes,  infects the cytoplasm of enterocytes in humans.  spp. from fishes differ from E. bieneusi\n  Nucleospora  that they possess spores with 8 to 12 turns in  the polar filament, whereas those of E. bieneusi  have  only 5 or 6. On the basis of these 33  distinctions and the morphological characteristics available to date, the intranuclear microsporidian found in both Atlantic lumpfish and Atlantic halibut should also be transferred to the genus  Nucleospora.  Despite the reestablishment of Nucleospora  and Enterocytozoon  as separate  genera, they are more closely related to each other than they are to other microsporidian genera. Docker et al. (1997b), therefore, proposed that Nucleospora retained in the family Enterocytozooidae. Nucleospora  salmonis  be  exhibits many of the  distinctive characteristics of the family Enterocytozooidae (Cali & Owne 1990; Desportes-Livage et al. 1996). In other microsporidia, polar tubes do not form until sporonts divide and the plasmalemma has thickened, whereas Nucleospora  and  form polar tube precursors (i.e., electron dense discs) prior to  Enterocytozoon  plasmodial division and thickening of the sporoginal plasmalemma. In addition, both Nucleospora  and Enterocytozoon  are polysporous but do not form sporophorous  vesicles or pansporoblastic membranes, and do not possess diplokarya in any stage of development. Nevertheless, after more detailed comparisons, Desportes-Livage et al. (1996) concluded that the morphology and development of these microsporidia are less closely related than originally supposed. Nucleospora  infections are usually associated with a concurrent neoplastic  condition involving massive lymphoproliferation, known as plasmacytoid leukemia (PL) in chinook salmon in British Columbia (Kent et al. 1990). The actual cause of PL is controversial. Laboratory transmission studies indicated that N. salmonis  may not be the  primary cause of all cases of PL ; i.e., PL was transmitted with tissue homogenates or filtrates in the absence of the microsporidian (Kent & Dawe 1990; Newbound & Kent 1991; Kent & Dawe 1993). Furthermore, Eaton & Kent (1992) described a retrovirus from fish with PL. 34  The most convincing studies suggesting a viral etiology for PL was conducted with tissues from fish with no sign of N. salmonis  infection, which was often the case in  pen-reared salmon that were examined in the late 1980's and early 1990's (Kent ML pers. comm.). However, in essentially all cases that were investigated in recent years, and in studies from other countries, N. salmonis  is consistently observed in the  proliferating plasmablasts. Therefore, it is possible that these morphologically similar lymphoproliferative disorders are actually different diseases caused by two different parasites. Studies with fumagillin (Hedrick et al. 1991b) and its analog TNP-470 (Higgins et al. 1998) support the microsporidian hypothesis. In these studies, both the parasite and the lesions were prevented by treatment with these antimicrosporidian compounds, which is in contrast to earlier experiments with fumagillin in the control of PL (Kent & Dawe 1993). The microsporidian has been maintained in lymphocyte cultures, and soluble fractions of these cultures stimulate uninfected cells to proliferate (Wongtavatchai et al. 1995a). Sensitive and specific polymerase chain reaction (PCR) tests have been developed for the detection of N. salmonis  based on rDNA sequence from the small subunit region  (Barlough et al. 1995) or ITS region (Docker et al. 1997b). Pathology. Heavily infected fish are anemic, with a packed blood cell volume as low as 5%. Typical of severe anemia in fish, affected salmon exhibit prominent pallor of the gills. Histological examination of affected fish reveals a proliferation of plasmablasts in essentially every organ, including the kidney, spleen, liver, intestine, pancreas and associated mesenteric fat, meninges, heart, skeletal muscle, skin, and eye (Hedrick et al. 1990; Morrison et al. 1990). In histological sections, the plasmablasts contain large, often deeply clefted or lobated nuclei, and prominent nucleoli. They have a moderate amount of finely granular, eosinophilic or amphophilic cytoplasm, and many of these 35  cells are mitotically active. The eye, spleen, and kidney are the primary organs affected (Fig. 21). In the eye, there is massive infiltration of plasmablasts into the periorbital connective tissue and ocular muscles. Kidneys exhibit prominent hyperplasia of the interstitium due to proliferation of the plasmablasts. Thickened basement membranes in the capillaries of the glomeruli are often observed. In severely affected fish, there is a perivascular infiltration of the plasmablasts within the liver, and the cells often proliferate within the sinusoids. The pericardium of the ventricle, atrium, and bulbus arteriosus of the heart may be infiltrated by plasmablasts, which form a thick cellular capsule that surrounds the heart. When the lower intestine is affected, there is massive proliferation of the plasmablasts in the lamina propria and in the submucosa, resulting in expansion of the intestinal villi. The microsporidian is very small and is identified by careful examination of nuclei of hemoblasts in histological sections (Fig. 22), in Gram-stained imprints (Fig. 23), or by electron microscopy (Fig. 24). In tissue sections stained with hematoxylin and eosin, the parasites appear as eosinophilic spherical bodies (2 to 4 pm) in host cell nuclei, surrounded by a rim of basophilic host cell chromatin. The Warthin-Starry stain combined with hematoxylin and eosin enhances the detection of the parasite in tissue sections (Kent et al. 1995b). In these preparations, prespore stages stain brown or black, and spores stain dark black. In one outbreak, the infection in pen-reared Atlantic salmon was associated with multiple focal lesions appearing mainly on the head. Histologically the lesions were characterized by massive fibroplasia in which the nuclei of proliferating fibrocytes were infected by the parasite. Transmission. Nucleospora  salmonis  is transmitted by cohabitation or feeding infected 36  tissues to fish in fresh water (Baxa-Antonio et al. 1992). Kent ML (pers. comm.) repeated these findings in the laboratory, but was unable to transmit the infection by cohabitation in sea water.  SUMMARY AND THESIS OVERVIEW In recent years there has been an increased research interest in fish microsporidia along with an increase in the economic importance of microsporidia in fish culture. This has lead to a better understanding of the pathogenesis and immune response of the host to the parasite. Furthermore, advances are being made in the development of effective chemotherapeutants for these microsporidia. In the past, fish microsporidia were assigned to specific taxa based mostly on morphological characteristics. Unfortunately, few useful morphological characteristics are present in fish-infecting microsporidia. The use of molecular systematics, along with interhost transmission experiments, has provided a better understanding of the taxonomic relationships of fish microsporidia, and I suggest that these tools be employed (when possible) as new taxa are proposed. Aquaculture of salmonids in British Columbia has continued to expand since the early 1980's. As the industry grows successful diagnosis and treatment of disease outbreaks is vital (Kent & Poppe 1998). Very little is known about the basic biology of Loma  salmonae  an important pathogen of salmonids {Oncorhynchus  spp.) from the  Pacific Northwest. Yearly, Pacific salmon farmers incur significant economic losses due to epizootics of L. salmonae. L. salmonae  This thesis was undertaken to contribute to knowledge on  transmission and viability, host susceptibility and immunology. In addition,  wild salmonid and nonsalmonid hosts of the parasite were examined. Findings in these areas will be presented in subsequent chapters. 37  tr*  Fig. 1. Pleistophora hyphessobryconis of neon tetra Paracheirodon inessi.  infection in skeletal muscle Scale bar = 14p.m  38  Fig. 2. Microsporidium zebrafish Brachydanio  sp. infecting the central nervous system of rerio. Arrows denote xenomas. Scale bar = 35 um  39  Fig. 3. Wet mount of a ruptured xenoma filled with spores of undescribed Loma sp. from the gill of ling cod Ophiodon elongatus. Scale bar = 12 jum  40  Fig. 4. Pleistophora hippoglossoideos in the musculature of plaice Hippoglossoides platessoides. Reprinted with permission of the author and publisher from Moller & Moller (1986)  Fig. 5. Heavy infection of Loma salmonae (white xenomas) in fresh gill of wild spawning sockeye Oncorhynchus nerka salmon from Babine lake, British Columbia, Canada. Reprinted with permission from M. Higgins  42  ^ 0  aft  c£  0  •s  ft  ^  /Si  Fig. 6. Wet mount of spores from an undescribed microsporidian from mountain whitefish Prosopium williamsoni. Arrow indicates large posterior vacuole. Scale bar = 10 urn  Fig. 7. Electron micrograph of phagocytic cell with prominent nucleus (N) and remnants of Pleistophora ovariae spore walls within a food vacuole (FV). Scale bar = 2 (am. Reprinted with permission of the author and publisher from Canning & Lorn (1986)  44  Fig. 8. Large cysts of Glugea anomala in a stickleback Gasterosteus aculeatus Reprinted with permission of the author and publisher from Moller & Moller (1986)  Fig. 9. Abdominal cysts of Glugea hertwigi denoted by arrows in rainbow smelt Osmerus mordax. Reprinted with permission of the author and publisher from Noga (1996)  10  tH^jv.  k  Figs. 10 & 11. Glugea stephani in winter flounder Pleuronectes americanus. Fig. 10. Heavy infection of cysts in the intestine. Fig. 11. Close up to show pebbled chalky-white appearance of intestine in the same individual. Reprinted with permission of the author and publisher from Moller & Moller (1986)  47  14  v  15  \  . % A  i  l  l  »  ::Y*. ...  Figs. 12 to 15. Various stages of microsporidian development represented by histological sections of Loma salmonae in coho salmon Oncorhynchus kisutch gills. Fig. 12. Young xenoma in secondary lamellae denoted by arrow. Fig. 13. Mature xenoma with few remnants of host cell nuclei denoted by arrow. Scale bar = 45 u m . Fig. 14. Proliferative inflammatory response denoted by arrows associated with infection in the primary lamellae. Fig. 15. Granuloma resulting from a ruptured xenoma with a few remaining spores denoted by arrow in macrophages. Scale bar = 10 jam  48  Fig. 16. Tetramicra brevifilum denoted by arrows in skeletal musculature of turbot Scophthalmus maximus. Reprinted with permission of the author and publisher from Matthews & Matthews (1980)  49  Fig. 17. Pleistophora ovariae in the golden shiner Reprinted with permission from G. Hoffman  Notemigonus  chrysoleucas.  Fig. 18. Sporophorocyts of Heterosporis finiki in the musculature of angelfish Pterophyllum scalare. Scale bar = 20 um. Reprinted with permission of the publisher from Michel etal. (1989)  51  Figs. 19 & 20. Fig. 19. Micrsporidium takedai in heart of sockeye salmon Oncorhynchus nerka. Scale bar = 4 mm. Reprinted with permission of the author and publisher from Canning & Lorn (1986). Fig. 20. M. takedai in musculature of rainbow trout Oncorhynchus mykiss. Reproduced with permission of the authors and publisher from Lorn & Dykova (1992)  52  Fig. 21. Reno-splenomegaly in chinook salmon Oncorhynchus infected with Nucleospora salmonis. K: kidney; S: spleen  tshawytscha  53  Figs. 22 & 23. Fig. 22. Nucleospora salmonis denoted by arrows in the nuclei of hemoblasts from the retrobulbar tissue of the eye of chinook salmon Oncorhynchus tshawytscha in histological section. Scale bar = 10 u,m. Fig. 23. Gram stain of the same tissue, shows spores in nucleus remnant and free denoted by arrows. Scale bar = 10 urn  54  Fig. 24. Electron micrograph of Nucleospora of a hemoblast. Scale bar = 0.25 urn  salmonis  in the nucleus  55  CHAPTER II Modes of Transmission INTRODUCTION Loma  is a microsporidian parasite infecting endothelial cells of  salmonae,  salmonids. Fish infected with L. salmonae  often exhibit pale gills with petechial  hemorrhages, inflammation, hyperplasia, and white cysts termed xenomas (Wales & Wolf 1955; Hauck 1984; Kent 1992). Outbreaks of L. salmonae Oncorhynchus  in Pacific salmon  spp. and rainbow trout have occurred in the Pacific Northwest (Hauck  1984; Kent et al. 1989), eastern United States (Markey et al. 1994), and Scotland (Bruno et al. 1995). Kent et al. (1995a) demonstrated that fish can be infected by Loma  salmonae  when ingesting infected tissues. Whereas details on the early development of Loma species are unknown, in microsporidia that have been studied in this respect, the spore extrudes a polar filament that pierces a gut epithelial cell and injects the parasite's sporoplasm. For xenoma forming genera such as Loma and Glugea,  the sporoplasm  eventually divides forming numerous spores within a hypertrophied cell (i.e. a xenoma) (Canning & Lorn 1986). Loma  salmonae  has been transmitted experimentally both in fresh and sea water  by feeding fish macerated gills containing spores (Kent et al. 1995a). Hauck (1984) suggested that gills may be infected directly by phagocytic uptake of pillar cells. However, the method by which Loma spp. spread within the host is unknown. It may be by infected macrophages, or by being free in body fluids (Hauck 1984). In the present study, experimental transmission of L. salmonae various routes: peros,  to chinook and coho was attempted by  intraperitoneal (IP), intramuscular(IM), and intravascular (IV)  injection, cohabitation of infected live and dead fish with naive fish, and placement of L. 56  salmonae  spores directly on the gills.  MATERIALS AND METHODS Fish husbandry. Fish were obtained from Rosewall Creek Hatchery, Fisheries and Oceans Canada, Vancouver Island, Canada. Strains of chinook were donated by the industry from Sea Springs Hatchery in Duncan, Vancouver Island, Canada. These hatcheries receive only well water and have no history of L. salmonae.  During  experiments all fish were held in flow-through pot tanks (41 L), mid-ovals (204 L) or deep-ovals (726 L) on fresh or seawater at the Pacific Biological Station (PBS) in Nanaimo, British Columbia. This station receives residential freshwater, which is then dechlorinated. Seawater is pumped in from a depth of 30 m, filtered to 25pm, and provided for use. Temperatures, unless specified otherwise, were kept at 14 to 16°C. All effluent from experimental tanks was chlorinated (10 ppm) before discharge to city sewage.  Preparation of infective Loma salmonae  spores. All suspensions were kept at 4°C  during preparation of infective material. Gills were first obtained from infected chinook at a seawater netpen site on the west coast of Vancouver Island. Gill tissue was scraped from each arch using a scalpel and suspended in dechlorinated fresh water. This solution was ground (level 1, 20 s at 4°C) using a Polytron tissue homogeniser (Brinkmann Instruments Co., Rexdale, Ontario, Canada) to create a slurry. The slurry was centrifuged at 2000 x r/for 10 min at 4°C. The pellet was resuspended in water and filtered through a wire mesh and then a 50 pm nylon screen. The filtrate was either centrifuged at 800 x gfor 45 min on a layered 34%/51% Percol (Sigma) gradient or centrifuged at 2000 x gfor 10 min on a 51% layer both at 4°C. The latter method required a 1:10 dilution of filtrate, but rapidly produced a pure product. Pure spores 57  were collected from the pellet and Percol layers. Spores were stored at 4°C until used. Number of spores were determined using a standard haemocytometer. Fish handling procedures. Fish were fed commercial salmon feed every other day, and anesthetized with tricaine methanesulfonate (MS-222) before handling. Fish were infected per os with a 16g needle tipped with 2.0 mm inner diameter tubing by either a gill tissue slurry (0.25 ml) or by pure spores (0.1 ml). A reservoir of fish infected with L. salmonae  was maintained at PBS from Aug 1995-1998. During sampling fish were killed  with an overdose of MS-222, and the first left gill arch of each fish was examined by wet mount for L. salmonae.  Remaining gills were placed in Davidson's solution (Humason  1979), processed using standard histological techniques, and examined. Sample collection. Fish heart, stomach, intestine, kidney, liver, spleen, and a section of dorsal muscle were also preserved for histology. Experiments were terminated at 56 d unless otherwise noted, to allow sufficient time for development of visible xenomas (Kent et al. 1995a). Exposure protocol. Various experiments, termed trials, were conducted as outlined in Tables 1 & 2. Loma  salmonae  spores were introduced directly into the stomach as  described above (fish hangling procedures). For anal gavage, a 22 g needle tipped with 0.58 mm inner diameter tubing was used. Various numbers of pure spores (Tables 1 & 2) in 0.1 ml innoculum were used for anal gavage, IP, IM, and IV injections. Intravascular injections were given using the ventral gill sinus or dorsal aorta. Trials were conducted using the above exposure methods as outlined in Tables 1 & 2. Controls had their left ventral fin clipped and were held in the same tank as exposed fish. In trial 8 (outlined in Table 2) controls were given Earle's Buffered Saline Solution (EBSS) and blood smears were collected at all sampling times from fish receiving IV injections. In trial 3, blood smears from 3 IV injected fish were made within 58  30 min of injection. In trial 4, pure spores (0.1 ml) were placed directly on the left gill arch of 10 chinook held out of water for 1 min. After 1 min, each chinook was dipped for 5 s in a rinse bucket before being placed in the holding tank. Controls were passed through the rinse bucket before being placed in the same tank. The same innoculum was used here as in trial 1. For trial 5 donor chinook were infected per os and held for 72 d at 12 to 14°C. Ten donor fish with clinical signs of L. salmonae  (anemia and petechial hemorrhages on  gills) were placed in a 204 L tank with 45 naive chinook (differentiated by size from infected fish). After exposure periods, sets of 10 recipient fish were removed (Table 1). Following 7 d cohabitation (i.e. 79 d post-exposure of donor fish), all donor fish were examined for L. salmonae.  Intensity of infection in donors was measured from the  average of 3 counts of the number of xenomas per x100 field of view (1.5 mm diameter) converted to mm . 2  For trial 6 donor chinook were infected as in trial 5, selected for clinical signs of L. and killed with MS-222. The same protocol was then followed as in trial 5.  salmonae,  For trial 7 donor chinook were again infected as in trail 5 except 12 donor chinook were killed, and placed in 4 acrylic containers (62 x 32.3 x 11.7 cm) at 3 container" . Water was pumped into these containers at 1.7 L min" . Two 8.5 cm high 1  1  baffles kept dead fish from blocking outflow of each container that fed into a smaller pot tank containing 10 naive chinook. Four tanks of recipient chinook were exposed to outflow from the containers for 24 h, 3, 5, and 7 d after which they were placed on uncontaminated flow for 56 d before being examined for L. salmonae. were examined for L. salmonae  All donor fish  as in trial 5.  59  Detection of spores in blood. Blood smears from trial 3 were air dried, heat and methanol fixed, and an indirect fluorescent antibody test (IFAT) was conducted to locate spores. Monoclonal antibody (MAB) for L. salmonae  developed using standard  techniques (Schots et al. 1992) was provided by F. Markham, Department of Pathology and Microbiology, Atlantic Veterinary School, Charlottetown, Prince Edward Island, Canada. The MAB (225 pl) was applied directly to blood smears and allowed to incubate at room temperature for 30 min. The smears were washed 3 times and incubated in phosphate buffered saline (PBS) at pH 7.2 for 10 min. Fluorescein isothiocynate (FITC) conjugated goat anti-mouse IgG antibody diluted in PBS (1:128) was counterstained with 1% Evan's blue (1:156) and applied to the smears. Smears were incubated for 30 min, washed 3 times and incubated for 10 min in PBS. IFAT slides were examined with a fluorescent microscope at x1000 using pH 9 mounting fluid (Difco).  RESULTS All exposure methods, except placement of L. salmonae  spores directly on the  gill (trial 4, Table 1), resulted in infection (Tables 1 & 2). When administered  peros,  spores were found within 6.5 h in histological sections of stomach in free material of the lumen and in association with epithelial cells (Figs. 25 & 26). Between 2.5 and 24 h possible sporoplasms were found in epithelial cells of the stomach and pyloric caeca. These stages consisted of a dark staining body (probably nuclear material) surrounded by a lighter-staining area. At 24 h epithelial cells of the anterior intestine contained these sporoplasms, and some sporoplasms were also associated with the lamina propria (Figs. 27-29). These structures were not found in controls or in fish sampled at 35 or 56 d. 60  Loma  salmonae  spores were detected in the blood by IFAT 30 min after fish  received IV injections via the dorsal aorta and up to 24 h after injections via the ventral gill sinus (Fig. 30). By 56 d all fish given IP injections had swollen kidneys and spleens, petechial hemorrhaging, and bloody ascites. This was not seen in fish infected peros  or  in controls. One fish given an IV injection had edema and hemorrhaging. Fish receiving IM injections of spores exhibited edema and a pronounced inflammatory response in muscle tissue at the site of injection by 35 d (infection shown after 56 d in Fig. 31). No inflammation was seen in controls. In trial 8 (Table 2) xenomas were first detected 35 d post infection in gill, heart and spleen. Xenomas were found most often in the gills followed by heart and spleen (Table 3). Xenomas were not found in intestine, liver, muscle, or stomach. Xenomas in donor fish (Table 1; trials 5 to 7) were completely opaque and filled with spores. Intensity mm" in donor fish for trials 5 to 7 respectively was: 0.5 (range 0.22  1.3); 2.4 (range 0.2-3.7); 2.6 (range 0.2-4.0) per x100 field of view. Few fish became infected during cohabitation with dead donor fish, and no fish became infected when receiving outflow from tanks containing dead donor fish (Table 1; trials 6 & 7).  DISCUSSION Oncorhynchus  spp. were susceptible to L. salmonae  administered by per os,  anal gavage, IP, IM, and IV injection, and by cohabitation with infected fish. Microsporidia such as Glugea,  Heterosporis,  Microsporidium,  and Pleistophora  infect fish species, have been successfully experimentally transmitted peros,  that by IP, and  IM injection, and by water-borne exposure (Lorn 1969; Awakura 1974; McVicar 1975; Leiro et al. 1994). Water-borne infections can result from direct immersion in a spore solution (Awakura 1974; T'sui et al. 1988), or by feeding a crustacean, previously exposed to spores, to fish (Awakura 1974; Olson 1976, 1981). Loma  salmonae  has  been transmitted peros  to chinook (Kent & Dawe 1994) and rainbow trout (Speare et al.  1998a). In addition Speare et al. (1998b) transmitted L. salmonae  per os and by IP  injection of semi-purified spores to rainbow trout. Hauck (1984) suggested phagocytic uptake of Loma  sp. by gill pillar cells as water passes through the gills. I was  unsuccessful in infecting fish by placing spores directly on gills for 1 min. Association of L. salmonae  spores with stomach epithelial cells, and possible  sporoplasms released from spores located within epithelial cells of the stomach, pyloric caeca, and anterior intestine, most likely indicate initial sites of infection. Spores discharging polar filaments may pierce gut epithelial cells, injecting infective sporoplasms. These sporoplasms might move through epithelial cells and enter the lamina propria, which is richly supplied with blood capillaries. It is generally believed that microsporidia that infect fish enter gut epithelial cells, where they develop or are then transported to their preferred site of development (Canning & Lorn 1986). These microsporidia might infect migratory cells such as histiocytes, neutrophils or macrophages (Weissenberg 1968; Canning & Lorn 1986; McVicar 1975). Matthews & Matthews (1980) proposed that macrophages act as a transport mechanism for Tetramicra  brevifilum  through the endothelium of turbot, or become infected themselves  during phagocytosis of the parasite in the lamina propria of the intestine. However, to my knowledge none of these hypotheses have been previously demonstrated experimentally. Further studies using in situ hybridization would demonstrate the site of initial infection and how subsequent spread of L. salmonae Docker et al. (1997a) developed a PCR test for L. salmonae,  occurs within the fish host. which would be useful for  this purpose. Autoinfection within fish-infecting microsporidia has been proposed but not verified (Lorn & Dykova 1992). The systemic distribution of Loma sp. xenomas and free 62  spores suggests that, as xenomas rupture, infective stages are liberated and move throughout the fish via the blood (Hauck 1984; Markey et al. 1994). My experiments, in which free spores injected into the blood resulted in xenoma formation in the gills support the hypothesis that autoinfection may occur in L. salmonae,  i.e. in natural  infections, xenomas may rupture in blood vessels and free spores may circulate and establish new xenomas. The high number of xenomas in the gills compared to other organs, regardless of the route of exposure, suggests that the gill endothelium is the preferred site of development of L. salmonae,  rather than a coincidental site due to  route of exposure or circulatory patterns. Transmission of L. salmonae  from infected to cohabitating naive fish in a flow-  through system indicates how readily the parasite may be transmitted. My results also suggest the parasite is more easily transmitted from infected live fish than from infected dead fish. Kano et al. (1982) noted that Heterosporis  anguillarum  spread to healthy eels  from infected eels in the same aquarium. During cohabitation in my study, naive fish may have ingested spores from ruptured or whole xenomas released from the gills (i.e. the secondary lamellae) of infected fish. Spores could also be liberated in fish urine when fish have kidney infections (Hauck 1984). My results demonstrate that L. salmonae IP, IM) and natural (e.g. peros,  is transmissible by experimental (e.g.  cohabitation) exposure routes. Infection by IV injection,  and distribution of xenomas throughout vascular tissue suggests that once a fish is infected, L. salmonae proposed L. salmonae  may spread within the host by autoinfection. Kent et al. 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"P  C  0  E  "S  ® s-S >  O  H  0 (D  ^  o 5E  i sCO CD  co "a o .E co u_  Table 2. Loma salmonae in Oncorhynchus kisutch. Infection of L. salmonae in coho from trial 8 exposed by various routes to 1.2 x 10 spores fish" (except controls). Fish were considered positive upon detection of the parasite in wet mounts or histological sections 6  M o d e of e x p o s u r e  Per  N o . of fish 16  os  Time examined  1  No. positive/ No. examined  2.5 h 6.5 h 24 h 35 d 56 d 2.5 h - 5 6 d  0/2 0/2 0/2 1/2 7/8 0/8  2.5 h 6.5 h 24 h 35 d 56 d 2.5 h - 5 6 d  0/2 0/2 0/2 0/2 3/4 0/4  2.5 h 6.5 h 24 h 35 d 56 d 2.5 h - 5 6 d  0/2 0/2 0/2 1/2 6/6 0/8  2.5 h 6.5 h 24 h 35 d 56 d 2.5h-56d  0/2 0/2 0/2 1/2 6/7 0/7  a  a b  a  Control  8  Intraperitoneal  16  Control  4  Intravascular  0  16 d  d  d  Control  Intramuscular  Control a b c d  8  16  8  Possible sporoplasms detected in alimentary canal Spores detected in histological sections of stomach Injection by ventral gill sinus Spores detected in blood by IFAT  66  CO  co £. CD  CM  _co CO  l  o c  5^ CD  c  l o  O  o  9  CO  O O  CVI  S  LO CD CM  c o  E  CD  CO CO  c  o  o  O  _CO c  o co  !n  c  CD  o  o c •-  ^1I"  T-  n  0  CL  T -  CO CD  CM N CO CO CM T - CO "3" CM •<*•  00  5 1 CD CD CO tT  CD N  ^  •4—'  c ® o  CD CD CO C L  c  _Og "co 0  5  CO CD CO CD  X  •5-jg o  o  ZS CO X ! CD ~ =S CO CO  b> ~  •6  CO  &  .| 2 ^ CO CO 3 3 o O  ^  ; o  CM  O) ^  LO  CO  CO  £ > -£ -Q  § 2  o  g  CO  LL  •£ 9-  N  OJ CO  N  CD CD CO C 0  2 ~ co  T3  "C  O  CO CD CO LO  sz  "5 i_  0 g  CO  c  CD V j  3 CO  S "i  ~  I— o  .=  CO  o  ^  c o  o 0  CD  l  as o  "s-  CO  CO.  0  CO  c  CO CO CO  67  Figs. 25 & 26. Loma salmonae in Oncorhynchus kisutch. Hematoxylin & eosin sections of L. salmonae spores in coho salmon infected per os and sampled within 6.5 h. Fig. 25. Spores in stomach material denoted by arrows. Scale bar = 12 Lim. Fig. 26. Single spore in association with stomach epithelial cells. Scale bar = 12 Lim  68  CO  E  CO  re  Q. "o c o c L . re ( /> o <o E v. re  w  •  2 a  «• O  J2  2 ° co Q. CD o o B CN CO c  £. 2  CO CN  SE  CO  o re OH a> co «• o C JZ E •<> / o o « CO  « "5  II  *  CO  ro  c =  —  o  X  —  •Q E  > o a>  ZL ro t o  re co ° II •*0 -» is • re a>_: ro P £ a5 -° ro S 1 •Eco 0 2 «s c a  .•  3 -4= CO </)  i< &  •- s  <o .E•- ro E™ o rooro Q) c •o cC oC 1 1  1 o  ro  « *• •£ § C *ro O -S  SI  c re o <o  CN  Q. O  £ -j si cro re n -  co o *1«  JZ  Tf  ™  « JZ  N  O)  E  c *i IE £  is a) CN +. o Is o -Q ~ T3 co  C  CD  re  t  CO  co a> re co i l S co re  69  Fig. 30. Loma salmonae  in Oncorhynchus  kisutch.  L. salmonae  spores detected  by IFAT in the blood of coho salmon within 24 h offish being injected with pure spores via the ventral gill sinus. Scale bar = 12 jum  70  Fig. 31. Loma salmonae in Oncorhynchus kisutch. Pronounced inflammatory response in coho salmon muscle after 56 d in association with intramuscular injection of L. salmonae spores. Hematoxylin & eosin. Scale bar = 6 um  71  CHAPTER III Viability Under Various Laboratory Conditions INTRODUCTION Storage and longevity of insect-infecting microsporidia has been researched extensively (Oshima 1964; Henry & Oma 1974; Pilley 1978; Fuxa & Brooks 1979; Teetor-Barsch & Kramer 1979). Some species can be stored under refrigeration, in frozen water, lyophilized, or vacuum-dried quantities, and even long term (>20 y) in liquid nitrogen (Maddox & Solter 1996). These species retain viability for a long time, and have been documented surviving for years in only 4°C Ringer's solution (Revell 1960; Kramer 1970). In contrast, very little is known about storage and longevity of fish-infecting microsporidia. Lorn (1975) reported capillary tubes as a convenient method for storing spores, although he did not address viability. Lorn & Dykova (1992) reported microsporidia surviving up to 1 y in water at 4°C. Amigo et al. (1996) studied viability of Glugea  stephani  from  commercial flatfishes (family Pleuronectidae) using extrusion  rates of spore polar filaments, and flow cytometry. Flow cytometry indirectly represents viability by measuring membrane integrity. Amigo et al. (1996) found that G.  stephani  spores were able to resist heating (60°C for 30 min) and freezing (-19°C for 24 h), and retained extrusion rates (28.6%) and viability (67.3%) up to 17 mon. However, no studies to my knowledge, have examined the effect of disinfectants on fish-infecting microsporidia. lodophor disinfectants (1.0% iodine) are commonly used in the aquaculture industry to disinfect equipment, surfaces, and fish eggs. Docker et al. (1997a) found L. salmonae  spores in the ovaries of sexually-mature chinook salmon.  Although it has not been demonstrated, spores on the outside or inside of eggs might infect newly emerging fry. 72  No studies have examined viability of L. salmonae  stored under various  conditions. Viability of spores was determined by exclusion of the dye methylene blue and percent extrusion of spore polar filaments. Viability was also measured by ability of spores to infect fish. To address the risk of L. salmonae  spores infecting fry, I examined  the efficacy of an iodophor to kill spores at various concentrations.  MATERIALS AND METHODS Assays of spore viability. Spores were isolated as described in chapter II. Exclusion of methylene blue was based upon protocol described by Hoffman & Markiw (1977). Their assumption was that live myxosporean spores did not absorb methylene blue. I tested this assumption with L. salmonae  spores by incubating spores in various  treatments (Tables 4 & 5), and then combining 37 ul of spore suspension with 18 ul of 0.25 % methylene blue on a slide. A total of 300 spores counted at x1000 within 10 min were categorized as clear (viable) or blue (non-viable). Spores were exposed to arbitrarily chosen temperatures (Table 4). Extrusion counts were done by combining 30 ul of the spore solution with 30 ul of 3.0%  H2O2  on a slide. The solution was allowed to incubate 15 min at room temperature  and 300 spores were counted and categorized as extruded (visible polar filaments) or not extruded. Viability was measured directly by infecting fish peros  (see Chapter II) after  spores were exposed to various treatments (Tables 5 & 6). Aged dechlorinated aquarium water (fresh water) and sea water were filtered through 20 um nylon mesh before use. All incubation times were at 4°C unless otherwise indicated. Viability Trials  in fresh  or sea  water  1&2  Spores were kept in EBSS (1% penicillin-streptomycin antibiotic, GibcoBRL 10 73  000 units ml" penicillin G; 10 000 pg ml" streptomycin sulfate) 24 h and 5 d 1  1  respectively before being resuspended in various treatments (Table 4; Fig. 32). Artificial sea water (1.025 specific gravity) was created using Forty Fathoms salt (Marine Enterprises International Inc, Baltimore, MA). Antibiotic (1.0% penicillin-streptomycin) was added to treatments of distilled water and artificial sea water. At 7, 37, 99, and 190 d 10 fish treatment" (Fig. 32) were infected at a dose of 1.3 x 10 spores. Fish were 1  6  kept on sea water for 56 d before being examined for L. salmonae.  Spore suspensions  for trial 2 (Fig. 32) were changed only on days of methylene blue viability measurements. Trial  3  Spore dosage was 1.0 x 10 spores ml" with 2 fish treatment" given 0.5 ml. Fish 6  1  1  were fin clipped to differentiate treatment (Table 5), and placed in the same tank on freshwater (10 to 14°C). Spore suspensions were not changed, and total spore number ml  -1  was measured from a duplicate tube.  Trial 4  Per os dosage was 6.3 x 10 spores ml" with 5 fish treatment" given 0.25 ml 5  1  1  (Table 6). Fish were kept in separate freshwater tanks. Spore suspensions were changed once week" . Trials 5 & 6 followed the same protocol as trial 4 but 10 fish 1  treatment" were used. 1  Freezing treatment. Purified spores and infected gills were frozen in various solutions, thawed, and given to fish peros  as outlined in Table 7. Gill arches were  removed from infected chinook obtained from the reservoir tank (see Chapter II) and examined at x100. Gills with a high number of xenomas (> 2.1 mm") were selected for 2  freezing. Gills were quick frozen by placement directly in the freezer (-20°C & -70 °C), and slow frozen using a Nalgene Cryo 1°C freezing container (Gibco) for at least 24 h at 74  -20°C. Material was thawed gradually on ice and either filtered with 40 urn Nytex screen (whole gills) or centrifuged at x 2000 g for 10 min (pure spores) to remove test solution. Gills were resuspended in dechlorinated water and prepared for per os infection of fish (see Chapter II). lodophor treatment. Fish were experimentally infected following the protocol of chapter II with modifications described herein. Gill tissue suspended in dechlorinated fresh water was ground using the Polytron tissue homogeniser to create a homogenate (speed 1, 30 s at 4°C). This homogenate contained only free spores, disrupted cells, and cartilage debris while intact xenomas or host cells were absent. The homogenate was divided among 3 centrifuge tubes, centrifuged at 2000 x g for 10 min at 4°C, and resuspended in either dechlorinated fresh water (control), 100, 150, or 200 ppm iodine (Ovadine; Syndel International Inc.) for 15 min at 4°C. The suspensions were centrifuged again at 2000 x gtor  10 min and resuspended in dechlorinated water. Total  iodophor treatment time was 25 min. Two trials were run as outlined in Table 8, with 20 fish treatment" . Total length and weight of fish were recorded at the termination of the 1  experiment, and abundance of xenomas was determined from wet mounts as described in chapter II. In addition, gills from fish that were negative for the infection by this method were preserved in Davidson's fixative, and examined using standard histological techniques.  RESULTS Viability, as measured by methylene blue exclusion, decreased in spores subjected to extremes in temperature or long term storage (Table 4; Fig. 32). All chinook (avg. 8.1 cm; 4.2 g) infected peros  in trial 2 were negative. By 190 d all spores  incubated in ambient sea water were dead (Fig. 32). Spores stored at 4°C retained a high percent viability as indicated by methylene blue exclusion and infectivity tests 75  (chinook avg. 21.2 cm; 106.2 g), but began to die by 10 d in fresh water and 5 d in sea water (Table 5). Extrusion rates of recently isolated spores was as high as 51.0% decreasing over time to 0.0% at 100 d and 95 d, in fresh and seawater respectively. (Fig. 33). Prevalence of infection in chinook (avg. 15.2 x cm; 33.7 g) remained high (Table 6) while intensity decreased over length of time spores were stored in fresh or seawater (Figs. 34 & 35). Loma  salmonae  spores stored in either fresh or seawater were still able  to infect fish after 95 d (Table 6). Attempts to infect chinook (avg. 12.8 cm; 17.1 g) with spores that had been frozen were unsuccessful (Table 7). All groups of fish receiving iodophor treatment, as well as the controls, developed L. salmonae  (Table 8). Fish that received parasites treated at the highest concentration  (200 ppm) showed reduced prevalence and intensity of infection, and detection of parasites was delayed compared to other groups.  DISCUSSION My results suggest that methylene blue exclusion overestimates viability and is not an accurate measurement of L. salmonae  viability. Fish did not become infected by  exposure to spores held 7 d at 10°C, although approximately 80.0% of spores excluded methylene blue (Fig. 32). Keeping spores under these conditions, without weekly changing of solutions, might have led to acidification and inactivation of spores. Microsporidian spore extrusion requires an extracellular pH shift that triggers an influx of monovalent ions into the spore (Pleshinger & Weidner 1985; Frixione etal. 1992). Calcium may also be involved: it is concentrated in the polaroplast and C a  2 +  mobilization is associated with swelling (Pleshinger & Weidner 1985; Leitch et al. 1993). Polar filament extrusion has been successfully stimulated by monovalent cations 76  (Frixione et al. 1994) and inhibited by removal of Ca , raising osmolarity, and use of D2O, ethanol, and Hg  2+  (Leitch et al. 1993; Frixione et a. 1997). Extrusion rate also  overestimates viability as not all spores that extrude a polar filament also eject the sporoplasm (Amigo et al. 1996). I found L. salmonae  spore extrusion rates decreased over time reaching 0.0%  after 100 d storage (Fig. 33). Amigo et al. (1996) also reported decreased extrusion rates for G. stephani,  although rates did not reach 0.0%. I was still able to produce  infections in fish using spores that exhibited no extrusion in hydrogen peroxide. The osmolarity of the host alimentary canal may induce L. salmonae hydrogen peroxide does not. Loma  salmonae  spores to extrude when  spores lasted longest in fresh water  although I did not determine the maximum time that spores remain viable. It is possible that spores may be viable for longer than 100 d, however, I did not detect infections in 1 trial at 100 d. The rapid decrease in intensity of infections produced by spores stored for 95 d or longer indicates a lower number of viable spores, and it is unlikely L.  salmonae  spores would be viable for a year as described for some fish-infecting microsporidia (Lorn & Dykova 1992). Spores of L. salmonae  are also killed by rapid or slow freezing in  fresh or sea water, limiting the methods of long-term storage available to a researcher. Although L. salmonae  occurs in ovaries of sexually-mature salmon, I do not know  whether the parasite can be transmitted through eggs. Even if spores are not transmitted within eggs, eggs are often contaminated with blood and fecal material during spawning, and may become exposed to spores through contaminated ovarian fluid. Oral treatments with fumagillin (Kent & Dawe 1994; Higgins et al. 1998) and quinine hydrochloride (Speare et al. 1998c) may reduce L. salmonae  infections in fish, but  currently there are no approved drugs available to the fish farming industry. Iodine is an effective disinfectant for most egg pathogens and has been widely adopted by the 77  aquaculture industry (Evelyn et al. 1986). For salmonid eggs, the normal treatment is 100 ppm for 10 min at pH 7.0. I found that iodine at 100 ppm reduced L. salmonae  infections and was most  effective at 200 ppm. Although infections still developed, treatment decreased intensity of infections, and delayed their detection. Kumagai et al. (1998) found iodophor treatment of Cytophaga  psychrophila  reduced live bacteria numbers from salmonid  eggs, but failed to kill all bacteria even at 1000 ppm iodophor. My treatment was applied to very heavily infected gill homogenates, and use of iodophores on eggs and ovarian fluid in a field situation might be more effective as number of spores and reactive organic matter would be lower. Fertilized salmonid eggs can tolerate concentrations as high as 200 ppm iodine, although they require a pH > 6.0 (Alderman 1984). Fish farmers will have to determine what concentration is acceptable for each species and stock as mortality of eggs due to iodophor toxicity varies with species, parental stock, pH, egg condition, and state of development (Alderman 1984; Fowler & Banks 1990, 1991). My results indicate that exclusion of methylene blue and extrusion rates of L. salmonae  spores decrease when subjected to extremes in temperature or long term  storage. The most practical method for evaluating spore viability is their infectivity in fish. Vital staining with methylene blue overestimates spore viability. I was unable to determine the maximum viability of L salmonae  spores but my findings suggest it may  be close to 100 d in fresh or seawater at 4°C. To prevent transmission of the parasite to progeny, I recommend that farmers avoid contamination of gametes with tissue products (e.g., ovarian tissue or blood), and continue to use standard iodophor disinfection protocols for eggs, using the highest concentrations that are non-destructive to eggs or embryos. 78  Table 4. Loma salmonae spore viability as measured by methylene blue exclusion for various treatments in dechlorinated (aquarium) fresh water in trial 1 Treatment  24 °C 24 °C 92 °C 97 °C -20 °C -20 °C -70 °C -70 ° C  a  Length of treatment  24 h 7d 10 min 10 min 1h 48 h 24 h 24 h  Solution was frozen, thawed, and refrozen  Percent excluding methylene blue 68.0 70.8 4.5 0.0 26.5 7.3 38.3 3.0  Table 5. Percent viability of Loma salmonae spores incubated in fresh water (aquarium) or sea water (ambient) for various times as measured by methylene blue exclusion, per os infection of chinook salmon Oncorhynchus tshawytscha, and changes in spore number in trial 3 Solution  Fresh water  Sea water  Incubation time  Percent viable (Methylene blue)  No. positive/No. examined  1h 24 h 3d 5d 7d 10d  85.0 84.0 82.0 82.0 83.0 84.0  2/2 2/2 2/2 0/2 2/2 2/2  1.0 1.0 1.0 1.0 1.0 7.0  x x x x x x  10 10 10 10 10 10  6  1h 24 h 3d 5d 7d 10d  83.0 83.0 83.0 78.0 80.0 82.0  2/2 1/2 2/2 2/2 2/2 1/2  1.0 1.0 1.0 8.0 7.0 7.0  x x x x x x  10 10 10 10 10 10  6  Spore number ml" 1  6 6 6  6 5  6 6 5 5 5  80  Table 6. Prevalence of Loma salmonae in chinook salmon Oncorhynchus tshawytscha infected peros with spores previously incubated in fresh water (aquarium), sea water (ambient), or Earl's Balanced Salt Solution (EBSS) over time for 3 separate trials Solution  Fresh water  Sea water  EBSS  Incubation time  No. positive/No. examined Trial 4  Trial 5  Trial 6  24 h 12 d 18d 50 d 78 d 95 d 100 d  4/5 3/3 5/5 5/5 -  10/10 10/10 0/10 0/10  9/10 9/10 10/10 8/10  24 h 12 d 18 d 50 d 78 d 95 d 100 d  _  10/10 10/10 10/10 NA  10/10  _  _  -  -  -  -  24 h 12d 18 d 50 d  -  -  5/5 3/3 5/5 4/5  -  10/10 10/10 10/10 -  - = Not determined NA = All spores dead  81  Table 7. Gill material infected with Loma salmonae was stored under various conditions and times before peros exposure to chinook salmon Oncorhynchus tshawytscha at 4.1 x 10 spores fish 6  Solution  DMSO DMSO Dechlorinated freshwater EBSS Dechlorinated freshwater DMSO Dechlorinated freshwater a  Temperature  -20 -20 -20 -20 -20 -20 -20  °C °C °C °C & -70 °C °C & -70 °C °C °C  Time  15 13 13 24 24 24 24  d d d h & 48 h h & 48 h h h  No. fish positive/No. fish exposed 0/12 0/20 0/20 0/5 0/5 0/3 0/3 b  b  8% dimethyl sulphoxide This trial used purified spores  a  b  82  CO CO £ >. Z5 _Q CO  T3 CD O CD  C CO T3 o  .2 E  s  0  C CD CD  g » C CD  o c E =5 "co .2 CO  ^  H_  o  O CO O £Z  c o o co c D CD cC CO O C c o o o CO  CD  CO  \  T3 O  T3  | g OT E o CO CL X . 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TJ CD -t—»  O CD  CO  o3 LO E o CT3  LO CO  "S O O  CO CO  LO CO  t l  o  CO  co  g CO D o C CL E ICO CO CO c CO o C O c  00  o m  ^  Q  o  c o c JZ o  CD CO c o  C ZiO  cs FJ  CO CO CO i_ C CD oC O O J= ^ CO CO c: cz CD —  LO CO LO CM LO CO CM T-  ^LULU  LO O  O  / s e i u o u a x  IP  CO * E  x o cu "w  £ CD  •ON "BAV  < s-  .9?  co  UL O  86  T3  3  O  3  CD  o0 LO CO CO •4—"  E o M—  CO  -c o Q  % CO  -c CO LO  CO  "CO  "CO  CO  o o  -c o 0 c E •1  0  o CO o  c O  LO CD  "D O  c  1  0  o CL E CO  00  O LO  > Q  CO c CO 'co CO 0 o ll o 0  c _c  1c  o 0  c !X CD c CO 0  Imo ami  c  CO — CO 0  oma wat  00  •~j co 0  "o CO >* " c —  o  co C o 0  c 4—•» CO  •ON "BAV  en  LULU / S B L U o u e x  CO T3 E 0 o O CO X CO 0 0 L_ CO 0 CO CL i_ CO 0 H—• > -SZ  <  LO CO CO LL  CO 0 0) CL.  CHAPTER IV Host Susceptibility INTRODUCTION Loma  salmonae  occurs mainly in species of Pacific salmon, although Poynton  (1986) described it in brown trout and Wales & Wolf (1995) in an unidentified Cottus Recently Shaw et al. (1997) described L. embiotocia  sp.  in shiner perch, and new Loma  spp. are being found in numerous marine fishes (Kent et al. 1998). Host specificity of L. salmonae  was first examined by Kent et al. (1995a) who unsuccessfully attempted to  infect Atlantic salmon, shiner perch, and Pacific herring. Further research has been limited to documenting natural infections of L.  salmonae.  Genetic variation between strains of fish from different geographic areas has been demonstrated in viral, bacterial, and protist infections. LaPatra et al. (1996) found hybrid strains of brown and lake trout Salvelinus  namaycush  Walbaum were more  resistant to infectious hematopoietic necrosis virus (IHNV) than were rainbow trout. Carp Cyprinus salmonicida  carpio  L. exhibit variable resistance to furunculosis  Aermonas  (Houghton et al. 1991), and Atlantic salmon, chinook, coho, and chum  salmon show variation in resistance to vibriosis Vibrio salmonicida, disease Renibacterium  salmoninarum,  bacterial kidney  and furunculosis (Withler & Evelyn 1990;  Beacham & Evelyn 1992; Gjedrem & Gjoen 1995). Clayton & Prince (1992) noted both inter and intraspecific variation in resistance to the ciliate Ichthyophthirius  multifiliis  Fouquet, 1876. Numerous studies have examined differential susceptibility of salmonid strains to the myxosporean Ceratomyxa Cryptobia  salmositica  shasta  Noble, 1950 and haemoflagellate  Katz, 1951 (Bower & Evelyn 1988; Ibarra et al. 1991, 1992, 1994;  Bower 1995; Bower et al. 1995; Forward et al. 1995; Forward & Woo 1996). Chevassus & Dorson (1990) and Wiegertjes et al. (1996) provide extensive reviews of genetic 88  variation in resistance to disease in fish. No studies have examined host specificity of L. salmonae  or strain differences  within chinook salmon. Therefore, I assessed host specificity of L. salmonae  with  selected salmonid species, and unrelated fresh and seawater fish species. A fish farming company had anecdotally reported different susceptibility of 3 chinook strains to L. salmonae.  This was investigated by infecting these strains under controlled  conditions.  MATERIALS AND METHODS Species susceptibility. Fishes were tested as outlined in Table 9, and all were tested using the following methodology except where noted. Twenty fish from a test species were placed in a tank (fresh or seawater dependent on fish species) with 10 positive control fish (chinook or coho), and taken off food for 2 d. Only 10 goldfish auratus  L. and 10 guppies Poecilia  reticulata  Carassius  were tested. Fish were fed macerated gill  tissue over 3 d. During feeding, water flow was turned off for 2 h to facilitate ingestion of gill tissue. On the fourth day 10 experimental fish and 5 controls were removed and infected peros  with a gill slurry (see Chapter II), fin clipped, and placed back in the tank.  Using per os exposure ensured at least 10 fish were exposed to the parasite. All fish were examined 56 d later by wet mount and histology for L.  salmonae.  When test species were non-salmonids and smaller than controls, the test species were kept in a separate tank during the feeding of macerated gill tissue. On day 4 after exposure all fish were placed in the same tank. Strain susceptibility. Susceptibility of 3 strains (named after egg source) of chinook was examined in 2 separate trials. Southern Coastal (SC) is named after wild stocks of Big Qualicum River, Vancouver, Island, Canada. Northern Stream (NS) is named after 89  the Yukon River, Yukon, and a hybrid (H) of these 2 strains. All fish were obtained from Sea Springs Hatchery, Duncan, Vancouver, Island, Canada. Before trials, 30 fish from each strain were screened by wet mount, and histology for infection by L.  salmonae.  Trial 1  Seventy fish strain" were fin-clipped, placed in separate deep-oval tanks (fresh 1  water, 18°C), taken off food for 2 d, and then fed 23.8 g of macerated L.  salmonae  infected gill tissue tank" over 3 d. On day 4 all fish were placed in a single tank to which 1  17 controls strain" were added. Length and weight were recorded 27 d later when fish 1  were examined for prevalence and intensity of L. salmonae Prevalence and intensity of L. salmonae  by histology only.  was also recorded for dead fish by wet mount.  Viscera from 20 fish strain" were preserved for histological examination. 1  Trial 2  A group of 120 fish strain" were pit tagged (BioSonics Inc., Seattle, WA), and 1  held in tanks for 4 wk to heal from tagging. One hundred fish strain" were given L. 1  salmonae  per os at 4.5 x 10 spores ml" , and placed in 1 deep-oval tank. A day later 10 6  1  controls strain" were added. Length and weight of fish, and prevalence and intensity of 1  L. salmonae,  was recorded in dead fish, and all fish after 43 d (50% mortality overall). At  43 d all fish were examined for the parasite by histology and wet mounts. Statistics. Prevalence data were tested using a Chi-square test for goodness of fit. Intensity data was log transformed X'=log(X+1), then tested with an analysis of variance (ANOVA), followed by a Tukey Honestly Significantly Different (HSD) test (Zar 1984). Histological and wet mount enumeration were compared as an estimate of abundance in trial 2. Time to death and survival was also tested for trial 2 first by ANOVA, and then by THSD test.  90  RESULTS Host susceptibility. All 5 non-salmonid species tested were negative, although controls became infected (Tables 9 & 10). All salmonid species tested became infected except Atlantic salmon and Arctic char. In most cases, a higher number of positives were obtained using the per os method of infection rather than feeding macerated tissue. Strain susceptibility. All pre-screened fish were negative. Average size of fish in trial 1 was: SC 11.0 cm, 11.6 g; NS 9.3 cm, 6.9 g; H 12.9 cm, 21.0 g. Average size for trial 2 was: SC 12.3 cm, 22.0 g; NS 9.5 cm, 10.0 g; H 11.6 cm, 18.0 g. There were no significant differences between strains for prevalence of infection, however, intensity was consistently significantly higher (p<0.001) in the NS fish (Table 11). One outlier was omitted in trial 1. In trial 2 the NS strain had significantly higher numbers of xenomas when screened by either histology or wet mount (Table 12). This was also true for mortalities (Table 13), although days to death did not differ significantly (p=0.152) between strains of chinook (Fig. 36). Survival in strain NS was significantly lower (p=0.0).  Loma  salmonae  was distributed in all organs sampled (Table 14; Figs. 37-40).  DISCUSSION The ability to infect chum and pink salmon verifies that L. salmonae 7 species of Pacific salmon. Anonymous (1984) recorded L. salmonae  can infect all  in gills and  somatic muscle of pink, chum, and coho from Kemano river, and rainbow trout from Kenny Dam area British Columbia, Canada. They identified spores from stained smears of somatic muscle suggesting fish were heavily infected. I rarely detected xenomas in sections of muscle (Table 14) and only in the dermis of heavily infected fish (Fig. 39). In 91  addition I was able to infect brook trout, which is in contrast to findings of Speare et al. (1998a). Brook trout used by Speare et al. (1998a) may have been previously exposed to L salmonae  or L. fontinalis,  and may thus have developed resistance to re-infection  (Speare DJ pers. comm.). In light of my ability to infect brook trout, L.  fontinalis  described by Morrison & Sprague (1983) may well have been L. salmonae. be investigated further using PCR primers for L. salmonae (1997a) on samples of L. fontinalis.  Loma  This should  developed by Docker et al.  spp. that are distinct tend to differ in their  ribosomal DNA sequence (rDNA). For example, Shaw et al. (1997) found that the internal transcribed spacer region of L. embiotocia salmonae  differed significantly from that of L.  (see Chapter VI).  Usually most fish are susceptible to initial infection but differ in their ability to limit or destroy the pathogen (Wiegertjes et al. 1996). The terminology "resistant" and "susceptible" therefore, is always relative. A reproducible method of exposure is vital in demonstrating differences between strains. My first trial involved feeding macerated gill tissue and larger fish may have consumed more gill tissue leading to higher intensity of infections. Hence, trial 2 where equal doses were given to fish, will be discussed herein. Intensity of infection was significantly higher and survival significantly lower in the Northern Stream (NS) strain compared to the other 2 strains. In addition, samples of wild Y fish screened for L. salmonae  (see Chapter VI) were negative suggesting this  strain may represent a naive strain. The northern distribution of NS chinook may exceed the natural distribution of L. salmonae,  or NS fish may be exposed to lower numbers of  parasite. Bower et al. (1995) found that coho salmon from the Kitimat River were highly susceptible to C. salmositica  compared with coho from Big Qualicum River. They  postulated this may be because the parasite and it's vector are not enzootic to the Kitimat River. 92  Natural disease resistance in fish is a complex interaction of genetic and nongenetic factors that may have multiple components (Chevassus & Dorson 1990). Price (1985) discusses genetic factors that include: barriers (i.e. skin and mucous); effects on pathogens of the host environment (i.e. gastric secretions, pH of blood, deficiencies in compounds required by the pathogen, natural antitoxins); and humoral and/or cellular immune responses. Non-genetic factors may include pathogen abundance, virulence, and temperature effects on pathogen development (Ibarra et al. 1994). Differences in disease resistance between strains of fish has been linked to variation in levels of mucus precipitin activity (Cipriano et al. 1994), plasma lipid components (Maita et al. 1998), antibody production (Stronsheim et al. 1994a,b), complement (Roed et al. 1990, 1992, 1993), and respiratory burst activity in head kidney macrophages (Balfry et al. 1994). I found that significantly fewer Nothern Stream chinook survived than SC or H and that there were no differences in time to death. Survivability is considered the best criterion for evaluating resistance in strains of fish as this represents the sum of all hostparasite interactions (Wassom & Kelly 1990). Ibarra et al. (1991) noted only 13% of the C. shasta  resistant strain of rainbow trout died compared with 90% of the susceptible  strain, although they did not find any difference in time to death (Ibarra et al. 1994). Studies of C. shasta  suggest two interacting mechanisms: initial invasion by the parasite  and the ability of the fish to mount an effective immune response (Ibarra et al. 1994). This may also apply to L. salmonae,  and possible immunological mechanisms  accounting for strain and species differences will be discussed in chapter V. The SC strain has been farmed for a much longer period of time than the NS strain and therefore, may be more susceptible to the stress of captive conditions. The host range specificity of L. salmonae  now includes all 7 species of Pacific 93  salmon (Oncorhynchus),  and brook trout. Atlantic salmon are resistant and brown trout  are susceptible indicating that L. salmonae's  host range cannot be strictly determined  by the generic status of the host. My findings of a naive strain of chinook likely demonstrate both genetic and non-genetic factors are involved in L.  salmonae  resistance. 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Histological section of a Loma salmonae xenoma denoted by arrow in a chinook salmon Oncorhynchus tshawytscha artery adjacent to pyloric caeca. Scale bar = 14 uim  102  Figs. 38 to 40. Histological sections of Loma salmonae xenomas in chinook salmon Oncorhynchus tshawytscha tissue. Fig. 38. Xenoma denoted by arrow in a section of intestine. Scale bar = 20 Lim. Fig. 39. Xenoma denoted by arrow in dermis of dorsal muscle section. Scale bar = 20 Lim. Fig. 40. Xenoma denoted by arrow in mesentery. Scale bar = 20 (am 1 (  CHAPTER V Immunology INTRODUCTION Although they are generally poorly understood, humoral and cellular responses to microsporidian infections occur in fish (Kim et al. 1996). Specific humoral immune responses have been reported in fish serum (Leiro et al. 1993, 1996a,b; Kim et al. 1996; Hung et al. 1997), and mucosal secretions (Hung et al. 1996). A first line of defense includes proteases, lysins, and agglutinins in mucosal secretions, followed by mucosal cells, blood cells, and finally the reticuloendothelial system, comprised of endocytically active cells such as endothelial cells, granulocytes, and macrophages (Dalmo et al. 1997). Phagocytes such as granulocytes, monocytes, and macrophages are believed to be important in microsporidian infections of fish (Secombes & Fletcher 1992). Macrophages are present throughout the animal kingdom and play a central role in nonspecific defense (Dalmo et al. 1997). Although macrophages recognize and attack pathogens, they may also act as a route of infection. In mice infected with Encephalitozoon  cuniculi,  spores are engulfed by macrophages but not destroyed due  to prevention of phagosome-lysosome fusion, and the microsporidia E.  cuniculi  replicates within the macrophage (Weidner 1975; Schmidt & Shadduck 1984). A similar mechanism was discovered in Glugea  hertwigi  by Weidner & Sibley (1985) and it has  been suggested macrophages may act as a transport mechanism for microsporidia in fish (Matthews & Matthews-1980; Canning & Lorn 1986). Thus, examining the phagocytic response in fish may lead to a better understanding of interactions between microsporidia and fish (Secombes & Fletcher 1992). Tissue reactions to L. salmonae  have been well described (Hauck 1984; Kent et 104  al. 1989; Speare et al. 1989), but immune responses of the host are unknown. I investigated the in vitro phagocytosis of L. salmonae  spores by a resistant species  Atlantic salmon and two susceptible strains of chinook (Southern Coastal and Nothern Stream). The effects of complement activity on phagocytosis were quantified and compared to uptake of Baker's yeast Sarccharomyces  cerevisiae.  MATERIALS AND METHODS Macrophage isolation. Macrophages were isolated following protocol of Secombes (1990) with modifications described herein. All blood was removed from fish using heparinized vacutaners (VWR) and centrifuged at 2000 x a; for 10 min at 18°C. Plasma was collected and frozen at -86°C until use. Base media was RPMI-1640 (Sigma) with phenol red, and 80 u ml" heparin (Sigma) unless indicated otherwise. All solutions and 1  tissues were kept at 4°C. Total length and weight of fish was recorded before the head kidney was aseptically removed and placed in 25 ml media. Head kidney was macerated and pushed through a wire mesh tissue separator (Sigma) while being rinsed with media. The filtered suspension was centrifuged at 500 x g for 15 min and resuspended in 30 ml media using a pipette and 5 s of vortexing. This was layered in 3 ml aliquots on 34%/51% Percol (Sigma) gradients, centrifuged at 800 x g for 30 min at 4°C and macrophages recovered from the interface. Macrophages were washed twice (500 x g 10 min), counted using 0.25% Trypan blue (Sigma), and settled for 90 min (18°C) at 1.0 x 10 cells ml" in Leighton tubes (GibcoBRL) containing 9x24 mm glass 7  1  coverslips. Macrophages were then washed twice with media (18°C) and incubated for 48 h and media changed every 24 h before phagocytic assays. Adherence to glass and preincubation for at least 48 h before an assay results in > 95.0% macrophage purity (Secombes 1996). 105  Spore treatments.  Loma  salmonae  spores were purified (see Chapter II), kept at 4°C,  and used within 1 wk. All centrifugation was at 2000 x gfor 10 min at 4°C and incubation during an assay was done at 18°C for 90 min. Plasma collected from fishes was thawed on ice and subdivided into 500 LLI aliquots. Four treatments were prepared: normal plasma, no plasma (media only), and heat and EDTA inactivated plasma. Two replicates were run for each treatment. Plasma was heat inactivated at 50 °C for 30 min. A final concentration of 0.04 molar EDTA was used to inactivate plasma. Spore number was adjusted to 2.5 x 10 ml" , 1 ml aliquots centrifuged, resuspended, and incubated in 6  1  individual treatments. Phagocytic assays. After incubation, spores were centrifuged, resuspended in 1 ml media, and incubated in Leighton tubes with macrophages. Macrophages were then washed twice with the basal medium (18°C), and fixed in 70% methanol for 24 h, dried, and used in IFAT assays. IFAT assays. The same protocol as described for blood smears (see Chapter II) was used on coverslips with fixed macrophages. A final wash with distilled water was added to the protocol, and slides read 24 h later with a fluorescent microscope. Percent phagocytosis (PP) was defined as the percent of spores phagocytosed, and enumerated by counting 300 random spores as either phagocytosed or not (bound to surface of macrophages). The average of two replicate counts was recorded. Phagocytic index (PI) was defined as the average number of spores engulfed per macrophage, and was enumerated by counting the number of spores in 100 randomly selected macrophages that had engulfed spores.  Yeast assays. Baker's yeast  Sarccharomyces  cerevisiae  (Sigma) was inactivated as  described by (Matsuyama et al. 1992) with modifications described herein. Yeast was heated at 100°C for 1 h in PBS, washed 3 times, filtered through 50 urn Nytex, and 106  stored at -86°C until use. Macrophages were purified as described previously, and yeast cells were incubated in the same treatments as described for L. salmonae  spores. Fixed  macrophages were stained with Wright's Modified Leukostat Stain (Sigma) and examined at x1000. Fish types. Atlantic salmon, and 2 strains of chinook Southern Coastal and Nothern Stream (see Chapter IV) were examined for PP and PI of L. salmonae yeast. Ten fish of each strain were used for L. salmonae  spores and  spores, and 5 of each strain for  yeast assays. Statistics. All prevalence data was tested using a Chi-square test for goodness of fit. Intensity data was log transformed X'=log(X+1), and then tested with an ANOVA. Percent phagocytosis was examined with a 3 x 4 contingency table with 2 factors: fish type, and treatment. A Chi-square test when applicable, was used to test for differences between levels of the factors as outlined in Table 16. Phagocytic index was square root transformed X'= VX+.05, and tested with an ANOVA, followed by a Tukey HSD test.  RESULTS Phagocytic assays. Macrophages used in assays were purified from large fish (Table 15). Loma  salmonae  spores were easily differentiated as bound or engulfed (Figs. 41 &  42) by macrophages. Bound or engulfed yeast cells were also easily separated by phagosome formation around engulfed yeast cells (Fig. 43). Atlantic salmon macrophages had significantly (p<0.001) higher PP and PI than chinook (Figs. 44 & 46; Tables 16 & 17). Incubating spores in normal plasma produced significantly (p<0.001) higher uptake than in the other treatments for all fish types. No plasma or heat treated plasma resulted in equally low uptake however, EDTA treated plasma resulted in significantly (p<0.05) higher PP of spores compared to the former 107  treatments. There were no significant differences between fish strains or species in PP and PI of yeast cells (Figs. 45 & 47).  DISCUSSION Percent phagocytosis is usually defined as the percentage of cells containing target particles (Leiro et al. 1995). This definition was not useful in my study as greater than 95.0% of cells did not contain target particles therefore, I enumerated the number of target particles phagocytosed. Leiro et al. (1995) found that phagocytic uptake is affected by temperature, time, and pathogen to phagocyte ratio (R). For turbot spleen cells ingesting Glugea  caulleryi  spores R had the greatest impact on phagocytosis. R  was optimal at 68:1 for PP and 85:1 for PI of G. caulleryi  spores. Secombes & Fletcher  (1992) reported optimal killing of bacteria by macrophages usually occurred at an R of 1:2 or 1:10. It is difficult to estimate the R value in my study as a final cell count was not done (i.e., not all cells settled at 1.0 x 10 ml" adhered to the glass nor survived the 7  1  initial 48 h before assays were conducted). After pilot experiments with various numbers of spores, I chose a value that was easily read by IFAT, and resulted in PI values of 1 or more. As PP is considered less sensitive than PI (Leiro et al. 1995), my discussion herein will focus on the latter. I found significantly higher uptake by macrophages of spores preincubated in normal plasma versus heated, no plasma, or EDTA inactivated plasma. My results also suggested a higher uptake of yeast cells preincubated in normal plasma, but this was not statistically significant. Engstad & Robertsen (1993) also found this relationship with yeast particles engulfed by Atlantic salmon macrophages. Higher uptake of particles by fish macrophages in normal serum is well established (Sakai 1984; Johnson & Smith 1984; Matsuyama et al. 1992; Rose & Levine 1992; Sakai 1992; Secombes & Fletcher 108  1992). Normal serum contains complement (C) which increases phagocytosis through opsonization. Heat inactivation of serum inhibits C activity due to the presence of heat labile C components in serum (Sakai 1992). In addition, incubation of opsonized particles with antisera to fish C3 negates any opsonizing effect (Secombes 1996). Divalent cation C a  2 +  is essential for phagocytosis (Secombes 1996) and EDTA binds  such free cations. Conflicting reports as to the effectiveness of C as an opsonin are a result of variation in pathogens and species examined (Secombes & Fletcher 1992). Bower & Evelyn (1988) demonstrated the importance of C in acquired and innate resistance of sockeye salmon stocks to Cryptobia  salmositica.  Lysis of the parasite was  mediated by C and triggered by activation of the alternative pathway. Although reports certainly suggest the presence of C receptors on salmonid macrophages, no receptors have been isolated or cloned (Dalmo etal. 1997). In my study, Atlantic salmon macrophages had significantly higher spore uptake than macrophages of chinook. Atlantic salmon are resistant to L. salmonae  (Kent et al.  1995a) and higher phagocytic ability might be one of many mechanisms contributing to resistance. The main sites of phagocytic activity in fish include the lymphoid organs (kidney and spleen), peritoneum, atrium of the heart, and gills (Leiro et al. 1995). Mucosal intraepithelial and free lymphocytic aggregations have been identified in fish (Kaattari & Piganelli 1996; Steinhagen & Jendrysek 1994), and macrophages also exist free in the blood. Therefore, spores are likely recognized when initially entering the alimentary canal and later during autoinfection (see Chapter II). Microsporidia spore surface antigens are first recognized by fish immune cells (Leiro et al. 1993; Kim et al. 1996) , and spores also have recognition domains for antibody (Pomport-Castillon et al. 1997) . Kim et al. (1996) proposed that spores of G. plecoglossi\r\  ayu gut epithelium  result in delivery of antigens by intestinal phagocytes eliciting the immune response. 109  Atlantic salmon may also have a higher C activity or use another non-specific defense such as C-reactive protein, which can act as an opsonin (Dalmo et al. 1997). Alternatively, L. salmonae  may have evolved a mechanism to evade or suppress initial  binding by macrophages of chinook. Bacteria have many mechanisms for avoiding phagocytosis. They can avoid opsonization with C by intracellular infection, or by producing capsules (Matsuyama et al. 1992; Sakai 1992). Leukocytolysins produced by bacteria extracellularly can prevent ingestion by macrophages, and bacteria can resist intracellular killing within phagolysosomes (Goren et al. 1976; Sakai 1992). Leiro et al. (1996a) suggested that microsporidia have developed mechanisms for avoidance or suppression of the host immune response. Little information exists on these mechanisms in fish beyond immunological suppression (see Immunology Chapter I). Leiro et al. (1996b) proposed the phagocytic response of turbot is modulated by G. caulleryHo  the extent that serum antibody responses are decreased.  Although I did not find differences between the 2 strains of chinook in phagocytic uptake of spores, there may be differences in respiratory burst activity. Respiratory burst activity is believed to be phylogenetically conserved (Dalmo et al. 1997), and this might account for strain susceptibility differences I found (see Chapter IV). In addition, L salmonae  may prevent killing by macrophages in G. hertwigi  (Weidner & Sibley 1985)  through prevention of phagosome-lysosome fusion. Further investigations should examine this possibility at the ultrastructural level. In contrast to my findings with L. salmonae,  Leiro et al. (1996b) found no  significant differences in PP or PI of turbot spleen phagocytes (macrophages) given C or antibody treated G. caulleryispores.  They suggested turbot serum might contain  enzymes with chitinolytic activity degrading the number of spore surface binding sites. In addition to activity of C varying by species and target pathogen, macrophage activity 110  may vary with tissue type and species assayed (Secombes & Fletcher 1992; Neumann & Belosevic 1996; Dalmo et al. 1997). My results demonstrate that generalizations about C activity cannot be made about in vitro phagocytosis of fish-infecting microsporidia. Southern Coastal chinook had significantly higher phagocytosis of EDTA treated spores than Atlantic salmon indicating EDTA treatment of plasma may have been ineffective. Alternatively, non-specific binding might have occurred via lectins on the surface of macrophages or in the plasma to carbohydrates on the spore surface. Lectinophagocytosis is used to describe this process (Ofek & Sharon 1988). Saggers & Gould (1989) reported "lectin like" receptors in macrophages from tilapia aided in the phagocytosis of bacteria. The authors inhibited phagocytosis by blocking with various sugars, and proposed a dual function CR3 receptor similar to that described on human macrophages (Ross et al. 1985). This receptor contains a lectin and C part that act independently. My results show that macrophages from both chinook and Atlantic salmon phagocytosed yeast cells quite well regardless of treatment. The yeast cell wall is composed of (3-glucans which stimulate the nonspecific defense of animals. Jorgensen et al. (1991) reported 50-60% of salmon macrophages phagocytosed glucan particles in a serum free medium. Engstad & Robertsen (1993) demonstrated that Atlantic salmon macrophages recognize (3-glucan through either a specific receptor or indirectly as the glucan binds C or other serum opsonins: i.e., fibronectin is an important component of salmonid serum. In addition, their heat and EDTA treatments caused marked, although not statistically significant, reduction in opsonizing ability of serum. This may have been an artifact of their small sample size. Leiro et al. (1996b) also found a significant difference in sodium m-periodate (a mild oxidizing agent) treated spores and suggested lectinophagocytosis may play an important role in phagocytosis 111  of microsporidian spores. Although chinook salmon (ML Kent pers. comm.) and rainbow trout (Speare et al. 1998b) develop resistance to L. salmonae  when re-exposed, the mechanism of  resistance is unknown. It may involve phagocytosis by macrophages and expression of antigens and/or aspects of the humoral defense system. Neutralization and C fixation antibody and cell mediated immunity all play an important part in acquired immunity (Hung et al. 1996). Kim et al. (1996, 1997) found that antibodies to intact G.  plecoglossi  spores are produced by ayu but do not play a protective role against infection. In conclusion, my results show that C opsonization of L. salmonae  spores leads  to a significant increase in in vitro uptake by macrophages of Atlantic salmon and chinook salmon. Uptake by resistant Atlantic salmon macrophages was significantly higher than chinook macrophages indicating a possible mechanism of resistance. Simple differences in C activity between species might account for this or more complex mechanisms may be involved such as suppression or evasion of phagocytosis by L. salmonae  in chinook salmon. To investigate this further ultrastructural studies and  respiratory burst activity should be examined. My results also indicate that lectinophagocytosis may be involved in binding of L. salmonae  spores by chinook. This  is not surprising, as such nonspecific binding has been proposed for other fish-infecting microsporidia.  112  Table 15. Average length and weight of Atlantic salmon Salmo salar and 2 strains of chinook salmon Oncorhynchus tshawytscha that macrophages were purified from for immunoflurescent (IFAT) and yeast assays Fish type  Atlantic Southern Coastal Northern Stream  IFAT assay Length (cm) Weight (g) 39.6 26.1 32.8  668.6 214.7 393.7  Yeast assay Length (cm) Weight (g) 35.2 27.0 34.9  546.0 253.5 480.7  113  Table 16. Macrophage percent phagocytosis (percent of spores engulfed) of Loma salmonae spores between various comparisons of Atlantic salmon Salmo salar, and 2 strains of chinook salmon Oncorhynchus tshawytscha. Spores were preincubated in the plasma treatments Plasma Treatment Normal None Heated EDTA  Atlantic X Southern Coastal 96.7X80.5* 10.9X7.7 7.2X6.7 19.3X34.0*  Interaction by fish type Atlantic X Southern Coastal X Northern Stream Northern Stream 96.7X78.4* 80.5X78.4 10.9X9.5 7.7X9.5 7.2X6.7 6.7X6.7 19.3X31.2* 34.0X31.2  *Statistically different (p<0.05) by Chi-square test  114  Table 17. Macrophage phagocytic index (average number engulfed) of Loma salmonae spores between various comparisons of Atlantic salmon Salmo salar, and 2 strains of chinook salmon Oncorhynchus tshawytscha. Spores were preincubated in the plasma treatments Plasma Treatment Normal None Heated EDTA  Atlantic X Southern Coastal 2.8X1.5* 1.0X1.0 1.0X1.0 1.0X1.2*  Interaction by fish type Atlantic X Southern Coastal X Northern Stream Northern Stream 2.8X1.6* 1.5X1.6 1.0X1.0 1.0X1.0 1.0X1.0 1.0X1.0 1.0X1.0 1.2X1.0*  *Statistically different (p<0.001) by Tukey HSD  115  Figs. 41 & 42. Loma salmonae  phagocytosed by macrophages of Salmo salar and Immunofluorescent (IFAT) fields with green L. salmonae spores and counterstained red macrophages. Spores were preincubated in normal plasma (Atlantic salmon) or no plasma (chinook salmon). Fig. 41. Spores phagocytosed by Atlantic macrophages denoted by arrow. Scale bar = 6 urn. Fig. 42. Spores phagocytosed by chinook macrophages denoted by arrow and unphagocytosed denoted by arrowheads. Scale bar = 6 um Oncorhychus  tshawytscha.  1 1 f i  Fig. 43. Saccharomyces cerevisiae (arrow) phagocytosed by macrophages of Oncorhynchus tshawytscha stained with Wright's Modified Leukostat Stain. 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H El  - C CO Q. CD  co o o >  E 1-  TJ CD l l l l l l l l  _  Q LU  CO  CO  rag CD  co  <q -c 3  CD o  i—  O CD 4=  co  X  c  o o c  8o  E  o §.§ CO E co ^  0 c  O  CO  CD  CD  co CD  E ^ "° O O TJ C o b  co c co -c ~ -a ° -E. p o co CO O CO CO CO c CO CO _ CD CD .CO £ >. CO CL CO ^CM CD p C  c  as E o  ©? _^  CO  1_  1_  TO « j» CO  °  l  l  IS"  E i= 12 3 CO c c co o  xapuj ojiAooBeLid  CD  j-  C  | g CD - = > co _, co  <  CO  «  . o £ LI_ < . E  CHAPTER VI Wild Salmonid and Nonsalmonid Hosts INTRODUCTION Species of Loma  have been described from freshwater and marine fishes  (Morrison & Sprague 1981a, 1983) and Loma salmonae  is an important pathogen of  salmonid fishes reared in fresh and sea water (Wales & Wolf 1955; Kent et al. 1989). Nonsalmonid fishes have also been suggested as potential reservoirs for infections of L. salmonae  acquired by seawater-reared salmon (Kent et al. 1995a).  Shiner perch are common in and around salmon netpen farms in British Columbia, some of which have experienced severe outbreaks of L. salmonae.  A species of Loma  was found in the gills of shiner perch that were being examined as part of a study of potential reservoir hosts for L. salmonae. from L. salmonae  This species of Loma can be distinguished  by host and by geographic, morphological, and molecular (rDNA)  characters. I, therefore, describe this microsporidian as a new species (published previously by Shaw et al. 1997; refer to preface). Wales & Wolf (1955) first described L. salmonae  from yearling rainbow trout in  California. Kent et al. (1998) described Loma spp. from 5 species of Pacific salmon caught during their seawater phase. Little information exists on abundance of L. salmonae  in Pacific salmon during their freshwater phase. I surveyed prespawning,  spawning, and smolts migrating to sea from 5 rivers and 1 lake in British Columbia, Canada for L.  salmonae.  MATERIALS AND METHODS Nonsalmonid collection.  Cymatogaster  aggregata  were collected from 3 localities off  Vancouver Island, British Columbia, a commercial dock at Tofino and a netpen salmon 122  farm at Eagle Bay (both in Clayoquot Sound 13 km apart), and a beach at Departure Bay, Nanaimo. Fish were collected by seine in Nanaimo, by angling at the Clayoquot Sound localities, and by dipnet at the fish farm. Fish were killed with MS-222 then stored on ice or frozen. Fish were weighed and measured (total length), and the first 3 gill arches on the left side of each fish were examined for the presence of the parasite. Gill material for spore and xenoma measurements and abundance was collected at all sites. Material for histology was collected in July for the Eagle Bay sample, whereas material for electron microscopy was collected from the Tofino dock sample. Material for molecular biology was collected at all sites and frozen at -70°C. Salmonid collection. Salmonids were collected as outlined in Table 20. Fish were killed with a blow to the head or overdosed with MS-222. The first left gill arch was removed and examined by wet mount. Total length and weight was recorded for smolts only. Prevalence was determined for prespawning and spawning fish at x50 using a dissecting microscope. Intensity was examined as described in chapter II except a subsample of the first left gill arch was taken for large (spawning) fish. Sockeye smolts sampled from Great Central Lake were also screened by PCR (as described by Docker et al. 1997a). Smolts left over after examination on 10/22/97 were held in ambient seawater for 1 y and re-examined for L. salmonae. Smolts examined on 5/8/98 were sorted into a 1997 y class (< 3.0 g) and fish 2 y or older (> 8 g). Measurements and abundance. Xenomas and spores were measured from wet mounts of fresh material. For size comparison, 30 spores of L. salmonae from seawater-reared chinook salmon (British Columbia) were measured under identical conditions (fresh). Prevalence was determined by examination of gills in wet mount preparations, except for the July sample from Eagle Bay, in which prevalence was 123  determined from histological sections. Intensity was determined by counting the total number of xenomas in all gill arches of the infected fish collected from Eagle Bay in September. Wet mount preparations for prevalence and intensity were examined at x100. Fish were separated into underyearlings (<= 1-y-old) and adults (> 1-y-old) based on frequency plots and biology (Hart 1973). Histology. Gills were removed from fish and placed in Davidson's solution for histology. Histological sections were prepared from these gills and stained with hematoxylin and eosin. Electron microscopy. Infected lamellae were fixed for 24 hr in 4% glutaraldehyde, then transferred to Millonig's solution for an additional 24 hr. Lamellae were postfixed in 1% osmium tetraoxide, embedded in epoxy resin, sectioned, stained with lead citrate and uranyl acetate, and examined with a transmission electron microscope. Polymerase chain reaction (PCR) and DNA sequencing. DNA was extracted from infected shiner perch gill tissue from all 3 sites by proteinase K digestion at 37 °C for 6 h (10 mM Tris, pH 8.0; 10 mM EDTA; 1% SDS; 150 mM NaCl; 200 Lig ml" proteinase K). 1  The suspension was extracted twice with a 50:50:1 ratio of phenol to chloroform to isoamyl alcohol (pci), and the DNA precipitated on ice with ethanol. The DNA was resuspended in TE, and stored at 4°C. Fragments of the above DNA were amplified by PCR with 2 sets of primers developed from the rDNA sequence of L. salmonae  in chinook salmon (Docker et al.  1997a). The first set, the forward primer Loma f (5'-ATTAGTGAGACCTCAGCC-3') and the reverse primer LS-2 (5'-ATGACATCTCACATAATTGTG-3'), are located in the small subunit (SSU) and large subunit (LSU) rDNA genes, respectively; they amplify a 627-base pair (bp) fragment in L. salmonae  which spans the internal transcribed spacer  (ITS) region. The second PCR assay used the reverse primer LS-2 and a second 124  forward primer, LS-1 (5'-CTGGATCAGACCGATTTATAT-3'), which was developed from the hypervariable ITS region in L salmonae  and amplifies a 272-bp fragment in this  species. Docker et al. (1997a) found the second PCR assay (LS-1/LS-2) to be highly sensitive, capable of reliably detecting as few as 0.01 L. salmonae  spores per 50 LII  PCR reaction (or 40 spores g" of chinook salmon gill tissue). The first set of primers, 1  Loma f and LS-2, was 3 orders of magnitude less sensitive for L. salmonae  under these  conditions. Gene amplification was accomplished using standard PCR buffer (Gibco BRL), 1.5 mM MgCI , and 0.2 mM dNTP (Saiki 1990). Each 50 LLI reaction contained 25 pmol 2  of each primer, 1.25 units of Taq DNA polymerase, and 0.6 |ig of genomic DNA. The reactions were run in a PTC-200 Thermocycler (MJ Research, Watertown, MA) for 35 cycles of 94°C for 45 sec, 53°C for 45 sec, and 72°C for 90 sec; these were preceded by a 3 min denaturation at 95°C and followed by a 10 min 72°C extension. The PCR product generated by primers Loma f and LS-2 in the Tofino shiner perch sample was sequenced using the Sequenase PCR Product Sequencing Kit (United States Biochemical, Cleveland, OH), according to the manufacturer's instructions, and in the other 2 samples by Taq terminator sequencing using fluorescent dye-labeled terminators on the 373 DNA automated sequencer. Internal primers also generated from L. salmonae  sequence were used to sequence 47 to 97% of the product  in both directions. The sockeye smolt samples from Great Central Lake (10/22/97 & 5/8/98) were screened by PCR using the same protocol as described for nonsalmonids except for differences described herein. Gill arches from the 10/22/97 sample were lumped into groups of 5 before digestion and DNA amplification. The samples from 5/8/98 were separated by year class and individual samples digested for 24 hr at 37°C. Amplification 125  reactions were run at 45 cycles of 95°C for 35 sec, 54°C for 20 sec, and 72°C for 1 min; these were preceded by a 1 min denaturation at 95°C and followed by a 7 min 72°C extension.  RESULTS Nonsalmonid host. DESCRIPTION Loma  embiotocia  (Figs. 48-53)  Spores (n=30) are 4.8 Lim long (4.0-5.0 jim) x 2.6 Lim wide (2.0-3.0 Lim) and xenomas (n=10) 0.13 mm (0.06-0.16 mm). Xenomas are found in endothelial cells of secondary gill lamellae. Spores (n=20) have 14-18 coils of the polar filament. Earlier developmental stages (meronts, sporoblasts) and small tubules are present. Sporoblasts are not highly vacuolated and the sporophorous vesicle formed before sporogony. The xenoma wall is smooth lacking complex invaginations into xenoma cytoplasm.  Taxonomic summary. Type host: Cymatogaster  aggregata.  Type locality: Eagle Bay, Clayoquot Sound, Vancouver Island, British Columbia. Site of infection: Secondary lamellae of gills. Distinguishing characters: Round xenomas in gill, spore size, polar filament count and rDNA sequence. Material deposited: Hapantotypes in histological sections, Canadian Museum of 126  Nature, Invertebrate Collection (Parasites), Ottawa, Canada; sequence information as GenBank accession number U78815. Etymology: Named after family name for shiner perch, Embiotocidae.  Ecology.  Loma  embiotocia  was detected in the gills of shiner perch from all 3  locations (Table 18). Shiner perch examined ranged from 6.1 to 15.5 cm in length and 2.8 to 58.1 g in weight, which represented 2 y classes (underyearlings, 6.1 to 9.4 cm; adults, 10.5 to 15.5 cm). Both year classes were infected with L.  embiotocia.  Prevalence was as high as 15.2% in underyearlings and 13.5% in adults (Table 18). Intensity of L. embiotocia  in under-yearling fish (n=12) from Eagle Bay (September),  ranged from 4 to 583 xenomas fish" .  ,  1  Remarks. Spore size, the formation of xenomas of the cell-hypertrophy type, location in the gill, and mixture of developmental stages randomly throughout xenomas place this parasite within the genus Loma  (Canning & Lorn 1986). Species of Loma show  extreme overlap in spore size, number of polar filament turns, and other characters (Table 19). Loma salmonae  embiotocia  resembles Loma  diplodae  Bekhti and Bouix, 1985 and L.  in having a smooth xenoma wall lacking invaginations of the wall  (interdigitated) into the cytoplasm, a low level of sporoblast vacuolation, and in the developmental timing of the sporophorous vesicle. Note that L. branchialis insufficiently known for comparison (Table 19). Loma embiotocia  is  is distinguished from  L. diplodae  by host and geographic location. Host and rDNA sequence separate L.  embiotocia  from L.  salmonae.  The PCR primers Loma f and LS-2, both developed from the relatively conserved regions of rDNA sequence of L. salmonae, infected with L. embiotocia  yielded positive assays for the shiner perch  from all 3 sites; the size of the fragment was approximately  the same as that from L. salmonae  (Fig. 54). Repeated assays with primers LS-1, 127  designed from the hypervariable ITS region of L. salmonae,  and LS-2 consistently failed  to amplify DNA from any of the infected shiner perch (Fig. 54). Of the 635 bp amplified by primers Loma f and LS-2 in the 3 localities of shiner perch, 564 bp were sequenced and consisted of 334 bp of SSU rDNA sequence, 45 bp of ITS, and 185 bp of LSU rDNA, although the exact boundaries of the ITS are not known. As the PCR results suggested, the SSU rDNA and LSU rDNA regions in L. embiotocia  were similar to those of L. salmonae,  differing by only 4 and 2 base  substitutions, respectively. All 6 substitutions were transitions, and all were found in the 3 different L. embiotocia  isolates. There were no differences between these isolates in  the SSU and LSU gene fragments, although multiple nucleotide signals were observed at 1 SSU rDNA site in the Nanaimo (position 101, Fig. 55) and Eagle Bay (position 317, Fig. 55) samples. These sites may indicate that several slightly different copies were present, either due to the presence of different L. embiotocia  isolates in the pooled gill  samples or to differences among dispersed members of the ribosomal gene array. To determine this, cloning and sequencing of individual repeats is required. In contrast to the SSU and LSU gene fragments, the short ITS region (45 and 37 bp, respectively) differed significantly between L. embiotocia  and L. salmonae  by having  3 indels, 4 transitions, and 1 transversion (Fig. 55). The lack of amplification with primer LS-1 in infected shiner perch from all 3 sites reflects these sequence differences, many of which are at the 3' end of the LS-1 primer site. The ITS region was identical among L. embiotocia  from different localities, except for 2 sites each with 2 nucleotide signals in  the Eagle Bay sample (Fig. 55). Salmonid hosts. Loma salmonae  was detected in wild salmonids sampled from 4  rivers and 1 lake (Table 20). A prevalence of 30.0% occurred in prespawning sockeye which had recently entered freshwater during their migration to reproduce. Intensity of 128  infection was quite high in some fish with large xenomas clearly visible to the naked eye. None of the sockeye smolts held on ambient sea water for 1 y were positive by wet mount examination. I was unable to detect L. salmonae  in newly migrating smolts  except by PCR (Table 20; Figs. 56-58).  DISCUSSION In species of Loma that infect gills, most descriptions were based on spore measurements taken from formalin fixed spores, or from sections of resin-embedded material (Table 19). This makes comparison of the size of my spores with those of other species difficult. The number of polar filament coils overlaps, and developmentally there are only minor differences between most species of Loma  (Table 19). Morrison &  Sprague (1983) realized that the structure of spores of L. salmonae  and Loma  morhua  contained no differences of taxonomic value, and these species were separated based on differences in hosts and habitats. These species may be valid, as transmission studies by Kent et al. (1995a) suggest that L. salmonae  is host-specific, i.e., marine  fishes, including shiner perch, were not susceptible to experimental infection by L. salmonae.  The ribosomal DNA sequence comparisons presented here (Fig. 55) further demonstrate that L. embiotocia,  is distinct from L. salmonae,  although the 2 species  appear to be very closely related. Intraspecific differences in SSU-rDNA sequence have been studied in a number of microsporidia and have been shown to range from 0.2 to 11.4% within the genera Encephalitozoon,  Variomorpha,  and Nosema  (Baker et al.  1995). The 1.2% difference (4 base pairs in 334) found between L. embiotocia salmonae  and L.  SSU rDNA fragments approaches the lower end of the observed range and  thus reflects a close relationship between these species. The relatively low level of 129  variation nevertheless is greater than expected due to intraspecific variation (Didier et al. 1995). Although comparison between Enterocytozoon  bieneusi  SSU rDNA sequence  generated by 2 different studies (Zhu et al. 1994; Hartskeerl et al. 1993) yielded differences as high as 1.73%, intraspecific variation in the SSU gene of microsporidia is generally believed to be negligible (Didier et al. 1995; Docker et al. 1997b). Similarly, the more substantial differences in the ITS region of L. embiotocia  and L. salmonae  also  surpass that expected due to intraspecific variation. ITS sequence differences between the 3 isolates of L. embiotocia  were minimal, and intraspecific variation in the second  ITS region of mosquitos, flukes, and lake trout have also been shown to be negligible (Porter & Collins 1991; Adlard et al. 1993; Zhuo et al. 1994). Even where intraspecific variation has been shown to be more substantial (e.g., Fritz et al. 1994; Vogler & DeSalle 1994; Didier et al. 1995; Docker et al. 1997b), it was not of the magnitude noted in this study between L.embiotocia  and L.  salmonae.  PCR assays used in the present study were useful both in detecting species of  Loma infections that were not readily observed in wet mount preparations (primers Loma f and LS-2) and in distinguishing between L. salmonae and LS-2). In chinook salmon infected with L. salmonae  and L. embiotocia  (LS-1  however, the assay using  primers Loma f and LS-2 was found to be several orders of magnitude less sensitive than that using LS-1 and LS-2. A more sensitive PCR assay for the detection of L. embiotocia  in shiner perch could be developed by replacing Loma f with an L.  embiotocia  specific primer in the region of the LS-1 site in L. salmonae.  Such an assay  could speed transmission and cross-infection studies of potential reservoir hosts. Kent et al. (1998) identified other potential reservoir hosts for Loma spp. which include Pacific cod, tomcod Microgadus chalcogramma  proximus  Girard, walleye pollock  Pallas, lingcod, and sablefish Anoplopoma  fimbria  Theragra  Pallas. Description of 130  the Loma spp. from these hosts is ongoing by AMV Brown at the University of British Columbia. I found a low prevalence of L. salmonae  in 4 Pacific salmon species examined,  although intensity was quite high in some fish. Kent et al. (1998) reported a maximum prevalence of 41.7% for Loma spp. in wild Pacific salmon sampled from seawater. Anonymous (1984) noted L. salmonae  in gills and somatic musculature of coho, chum,  and pink salmon of the Kemano river and rainbow trout of the Kenny Dam area, British Columbia, Canada. They reported a maximum prevalence of 17% and considered the parasite to be typical of post-spawned fish in the Kemano river. Mortalities in spawning sockeye salmon from the Fulton river, British Columbia, Canada have been associated with L. salmonae  (MJ Higgins pers. comm.).  I detected fully developed infections in fish that had recently entered freshwater, and in spawning fish. Loma salmonae  was also present in smolts migrating out from  Great Central lake. It seems likely that smolts became infected by conspecific freshwater salmonids or by spores deposited with eggs (see Chapter III). I was unable to detect L. salmonae  xenomas by wet mount preparations after holding sockeye smolts  with subclinical infections (i.e. positive by PCR) for 1 y on ambient seawater. An appealing hypothesis is that fish first become infected as aelvins by spores deposited with eggs. Infected fish may maintain the infection subclinically as they mature during their seawater phase. Shortly before beginning their return migration, and when entering warmer water, fish may begin to develop clinical signs of L.  salmonae.  In conclusion, based on host, geographic location, spore morphology, and rDNA sequence, the microsporidian of shiner perch described here is a new species within the genus Loma. Loma,  To clarify the relationships and validity of other described species of  further comparisons should be done of inter- and intraspecific differences in 131  rDNA combined with transmission studies for members of the genus Loma. found L. salmonae  In addition I  to be prevalent in prespawning, spawning, and young (smolt) salmon  from rivers and lakes of British Columbia. This suggests that L. salmonae  may be  present throughout the life cycle of Pacific salmon maintaining itself subclinical^ until salmon mature at sea.  132  TJ C ZJ O i _  CO CO 3 'co CO  I00^  ICO  o  CO CM OJ  Tt  o  s  -I—'  CO TJ  CO o  0  O  "o O •2 CO CD  CD O c  8,  CO CD CO CO  _0  CO > CD  CO  I  s  LO  CM CO  O  I -; s  1—  LO OJ  CO OJ  CO CO LO  o CO o  o CO  LO  o 00  oo  o  o  CD  CD  T—  CO Zi TJ •«—'  <  CO CO c CO CD CD TJ c 3  o *  CD CL  ^ 0  £Z JZ CO c TJ c Zi o .CO o o  CO 0  00  E  0  CO CO  ZJ  M—  o  •S CD t CD CD T CO C s CO  £  CO CM  c o  CO Zi  <  a a a <0 0 0 " CO CO CO O  c CO 0  CO  —  o O 'to -c CO .t2 CO CQ c O TJ C O CO  Zi  ~3  0  CO  CQ  _0  CO CO  LU  CO  o o  o  0  o c  'co c CO  0  Q  £  1—  CO i_ CO CO Zi TJ  < (0  . 0 00 >  0  §  JZ) c CO CO H >  133  CO  C  -  s i  0  '  5  CD  >  | 8E O 2  0  C  JO  X! 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CO  0 CL  E  DC  as  O  CO  DL  L.  >>  CD CL CT5 CO CO CD h -  Q  o co co CM  E o ro  a CD  JQ 0 >  T3 CD CL  05  CO  E  1  o CL  _Z5 CD  •a  CD  CO X  i_ CD >  C  ir  JZ CO  E o ro  co E CO  CO CO  ® 0  CD  co  •£2 - >s  C?DC  in  00 CD  o  CD  CL  CO  is  "^S CM 3 00  •s  s  o r-ao  o ch o c >>  C CO > c bcb  Q  CO CO  00  •c o  § o  I  I  o o c  o  -c o  o u c O  5 a,  CO X  I  C  00 •3  f o o c  o  S  CD  0  m -c o  >  co J= co CD  CO  .00  CO J D  co OJ Z CO  CO  S  ~  I I  LO  a  -C CJ  ~  c  CD  cc 0  c  CO J Z CO  0 > ^ 0 i CO > CO 0 Q_ 0 o 0 II  CL CL  a  136  Fig. 48. Fresh spores of Loma embiotocia immersion. Scale bar = 10 um  observed under oil  137  Fig. 49. Xenoma of the hypertrophic cell type formed by Loma embiotocia associated with shiner perch Cymatogaster aggregata gill secondary lamellae. N; host cell nucleus remnants. Scale bar = 10 Lim  138  Figs. 50 & 51. Transmission electron micrographs of Loma embiotocia spores and xenoma structure. Fig. 50. Mature spore with 17 coils of the polar filament denoted by arrow. Scale bar = 1 um. Fig. 51. Small tubules denoted by arrow within xenoma cytoplasm. Scale bar = 1 um  139  Figs. 52 & 53. Transmission electron micrographs of Loma embiotocia spores and xenoma structure. Fig. 52. Sporoblasts (Sb) with minimal vacuolation within sporophorous vesicles (Spv). Scale bar =1 (am. Fig. 53. Smooth xenoma wall (W) containing spores (S). Scale bar =1 Lim 140  Loma f/LS-2  LS-1/LS-2  Fig. 54. PCR of DNA from chinook salmon Oncorhynchus tshawytscha gill infected with Loma salmonae and from gills of shiner perch Cymatogasteraggregata infected with L. embiotocia. Shiner perch were collected at Eagle Bay, Tofino Dock, and Nanaimo sites, and parasite DNA amplified with primers Loma f and LS-2 or primers LS-1 and LS-2 (1% agarose gel, ethidium-bromide stained). Negative control used distilled water instead of template DNA. Molecular weight markers (bp) are shown in the left lane  141  L. L. L. L.  salmonae embio T o f i n o embio EagleB embio Nanaim  CAGGTGCGCA CAGGTGCGCA CAGGTGCGCA CAGGTGCGCA  AAGCACAGGA AAGCACAGGA AAGCACAGGA AAGCACAGGA  AGGATGGGTC AGGATGGGTC AGGATGGGTC AGGATGGGTC  AAGGACAGGT AAGGACAGGT AAGGACAGGT AAGGACAGGT  CAGTGATGCC CAGTGATGCC CAGTGATGCC CAGTGATGCC  L. salmonae CTTAGATGGT L. embio T o f i n o CTTAGATGGT L. embio EagleB CTTAGATGGT L. embio Nanaim CTTAGATGGT  CCGGGCTGCA CCGGGCTGCA CCGGGCTGCA CCGGGCTGCA  CGCGCACTAC CGCGCACTAC CGCGCACTAC CGCGCACTAC  AGTGGTCGCC AGTGGTCGCC AGTGGTCGCC AGTGGTCGCC  GAAATTACCT GGAATTRCCT GGAATTRCCT GGAATTRCCT  L. salmonae GATAATTATA L. embio T o f i n o GATAATTATA L. embio EagleB GATAATTATA L. embio Nanaim RATAATTATA  AAGGCGATCG AAGGCGATCG AAGGCGATCG AAGGCGATCG  AGAGGGAATG AGAGGGAATG AGAGGGAATG AGAGGGAATG  AGCTTTGTAA AGCTTTGTAA AGCTTTGTAA AGCTTTGTAA  GAGGCTCAGG GAGGCTCAGG GAGGCTCAGG GAGGCTCAGG  L. salmonae AACGAGGAAT L. embio T o f i n o AACGAGGAAT L. embio EagleB AACGAGGAAT L. embio Nanaim AACGAGGAAT  TGCTAGTAAT TGCTAGTAAT TGCTAGTAAT TGCTAGTAAT  CGCGGACTCA CGCGGACTCA CGCGGACTCA CGCGGACTCA  TTAAGACGCG TTAAGACGCG TTAAGACGCG TTAAGACGCG  ATGAATACGT ATGAATACGT ATGAATACGT ATGAATACGT  L. salmonae CCCTGTTCTT L. embio T o f i n o CCCTGTTCTT L. embio EagleB CCCTGTTCTT L. embio Nanaim CCCTGTTCTT  TGTACACACC TGTACACACC TGTACACACC TGTACACACC  GCCCGTCGTT GCCCGTCGTT GCCCGTCGTT GCCCGTCGTT  ATCGAAGATG ATCGAAGATG ATCGAAGATG ATCGAAGATG  AAGATAGGCG AAGACAGGCG AAGACAGGCG AAGACAGGCG  L. salmonae CGAACGATCT L. embio T o f i n o CGAACGATCT L. embio EagleB CGAACGATCT L. embio Nanaim CGAACGATCT  ACCAGAAAGT ACCAGAAAGT ACCAGAAAGT ACCAGAAAGT  GAGCGCAGGT GAGCGCAGGT GAGCGCAGGT GAGCGCAGGT  TTTTAGATCT CTTTAGATCT CTTTAGATCT CTTTAGATCT  GATACAAGTC GATACAAGTC GATACAAGTC GATACAAGTC  L. salmonae L. embio T o f i n o L. embio EagleB L. embio Nanaim  AGCTGTAGGA AGCTGTAGGA AGCTGTWGGA AGCTGTAGGA  GAACCTGTAG GAACCTGTAG GAACCTGTAG GAACCTGTAG  CTGGatcaga CTGGatcaga CTGGatcaga CTGGatcaga  ccga ccgacaaaaa ccgacmaaaa ccgacaaaaa  GTAACAAGGT GTAACAAGGT GTAACAAGGT GTAACAAGGT *  *  *  +  + *  *  *  *  +  L. salmonae tttata L. embio T o f i n o g a a a c t c a t g L. embio EagleB g a a a c t c a t g L. embio Nanaim g a a a c t c a t g  taatctttgt t-atatt-at t-wtatt-at t-atatt-at  atgaatgtaa atgaatgtaa atgaatgtaa atgaatgtaa  tCTCTGCGCA tCTCTGCGCA tCTCTGCGCA tCTCTGCGCA  AGGGATCTTT AGGGATCTTT AGGGATCTTT AGGGATCTTT  L. salmonae TGGTTCGCTA L. embio T o f i n o TGGTTCGCTA L. embio EagleB TGGTTCGCTA L. embio Nanaim TGGTTCGCTA  GACGAAGAAG GACGAAGAAG GACGAAGAAG GACGAAGAAG  GGCGCAGCGG GGCGCAGCGG GGCGCAGCGG GGCGCAGCGG  AATGCGAAAT AATGCGAAAT AATGCGAAAT AATGCGAAAT  GTGCAGGAGT GTGCAGGAGT GTGCAGGAGT GTGCAGGAGT  L. salmonae CGCAGCGAAG L. embio T o f i n o CGCAGCGAAG L. embio EagleB CGCAGCGAAG L. embio Nanaim CGCAGCGAAG  ATAGCACATG ACAGCACATG ACAGCACATG ACAGCACATG  CCTGAAATCA CCTGAAATCA CCTGAAATCA CCTGAAATCA  CGAGAGTGAG CGAGAGTGAG CGAGAGTGAG CGAGAGTGAG  ACTACCCCTT ACTACCCCTT ACTACCCCTT ACTACCCCTT  L. salmonae TGAATTAAGC L. embio T o f i n o TGAATTAAGC L. embio EagleB TGAATTAAGC L. embio Nanaim TGAATTAAGC  ATATGAGTAA ATATGAGTAA ATATGAGTAA ATATGAGTAA  AGGGAGGAAA AGGGAGGAAA AGGGAGGAAA AGGGAGGAAA  AGAAACTAAC AAGGATTCCT AAAAACTAAC AAGGATTCCT AAAAACTAAC AAGGATTCCT AAAAACTAAC' AAGGATTCCT  L. salmonae GTAGTAGCGG L. embio T o f i n o GTAGTAGCGG L. embio EagleB GTAGTAGCGG L. embio Nanaim GTAGTAGCGG  CGAGCG CGAGCG CGAGCG CGAGCG  Fig. 55. Sequence alignment of the gene fragments sequenced here, including a portion (334 bp) of the small subunit ribosomal DNA (SSU rDNA) and part (185 bp) of the large subunit DNA (LSU rDNA), and the internal transcribed spacer (ITS), for Loma salmonae and Loma embiotocia (GenBank U78815). Lowercase letters denote the presumptive ITS. Differences between L. salmonae and L. embiotocia are marked with "*". Differences between L. embiotocia from different localities are marked with +. R indicates both G and A, W indicates both A and T, and M indicates both C and A  142  o C D  c o 1  o  *J  4 5 6 7 8 9 10 11 12 13 14 15 16  Fig. 56. PCR of DNA from sockeye salmon (Oncorhynchus nerka) gill infected with Loma Parasite DNA amplified with primers LS-1 and LS-2 (2% agarose gell, ethidium-bromide stained). The negative and positive controls (Ctr), respectively, used distilled water and DNA from purified L. salmonae spores. Lanes 5 to 16 are grouped samples of 5 fish per lane. Fish were collected at Great Central Lake, Vancouver Island, British Columbia, Canada as migrating smolts (1996 y class). Note positives in lanes 6, 8, 10, 12 & 15. Molecular weight markers (bp) are shown in lane 1 & 4 salmonae.  143  CL  O  o  _Q  If) T -  o o  a  o o o o  CO CO  'to  «* CO f  T—  co  •  o CO mi  CD CN 00 CN  CN CO CM CN CN  00  ff• A  CO  CN  C0  •  CN CO  m  to  1  1  gr  t  t  t  i  w  CO  #•  CO  o  CO CD  oo  •  CO T—  1  in  CM  3  T— T—  K  f  cn  in CO  00  CN  2  •  5 O O C  co in  o CD CO 00  Ti-  co  ro  hCO CO CO  '  1  m CO  3 O  —  c co  CO TJ O TJ CO CO c CO CD  i  i  O E  CO  >» co" o  iCO  > re  O  CN  »  to  >^ 0)  C J re cu — C  (0  u  SS  co re CCOL CL i : c co c o c c u c C ^u cO o CO  TJ co  O —  co V  r-  TJ  3 ^  o m  CO  CO  _  CO >  •C  m  ^ -  T—  1  c  in  —  )  I) >  CO  CO  TJ CO  re TO5 co E iS .2 * re CO TJ S I co  CD  T—  hin  Q)  CN  { 1  TJ  CO  m  *  T—  o  TJ *->  in 00 in  CO  iE  -J ^ t: co  CO  ft 1  . re  i i rec 82 1% c := 5 re -c co" II u CQ — 2 £  m  CD  CO  CQ CU  CN CO  in in in m  CO CO CO  c  c > -J re CO o += TJ -a c « > -S co cu re 'co co cu o V) c Q.  co CO  I t  CO  •a c C  CtJ CU  h~ CO CO CO  CO  CN O CN  T  m  >_  CO CO  CN CO CN  00  CO  JC co S o CO cu CO  in  to «  w  CO TJ  to Q. j O -Q 3  - cu o E 2 ccuo c=u CD ^ CU re o C D E ~ cu C CO >- to — CU o O JZ c s 9  to i-  re E ^ »  £ |  Q. 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CCOO £ 0 TJ ro 'E CD CD co 1 CD > CD *re E c t ci ™ S?g o ro co £ :  LO  0  c  < o ™I Iro  J»0JJ3 aeuoujies  a E  Z  7  Q  °<  _  -D  o o 10 T—  O O  co  o o  CO  ro o  co O |5  .S re T J "D  0 CD E o t 3 0) LO CO 'ro J o o LL CL 4-> CO Q. « ro U S .  a  CD JZ  CD  00 —  . ro  CHAPTER VII Conclusions and Future Recommendations  Microsporidia of fish can cause morbidity (e.g., clinical disease, decreased fecundity, swimming ability), mortality, and reduced marketability in wild and cultured fishes. Cultured fishes are particularly susceptible to infection because of high stocking densities used during rearing. Fish-infecting microsporidia can be broadly classified into two morphological groups: xenoma and non-xenoma forming. Both groups cause significant pathological changes and result in a host immune response. However, microsporidia can evade detection by the host's immune system due to intracellular sequestration. These parasites can also immunosuppress fish as seen with Glugea  spp.  and although antibodies are produced in some cases, it is unclear if they play a protective role. Presently, fumagillin and its analogue TNP-470 are the most promising drugs available for treatment. Although not currently licensed for use in fish-farming, both drugs control fish-infecting microsporidia, such as L. salmonae,  at low doses (2-4  or 0.1 mg kg" fish" day" respectively). If licensed these drugs may provide a treatment 1  1  1  to fish-farmers in the Pacific Northwest. In the mean time, a natural treatment for L.  salmonae  and other fish-infecting microsporidia may be manipulation of water  temperature. Growth of these parasites can be accelerated or retarded with raising or lowering of water temperature respectively. This thesis examined the basic biology of L. salmonae  in Pacific salmon and  related salmonids, and elucidates: mechanisms of parasite transmission; viability under laboratory conditions; host susceptibility; immunology; and potential wild and nonsalmonid hosts.  Modes of Transmission.  Loma  salmonae  is transmissible by exposing  Oncorhynchus  146  spp. per os to macerated gill or a gill slurry and by IP, IM, or IV injection of purified spores. Cohabitation of naive fish with infected dead or live fish in a flow through system will also transmit the parasite. Placement of infective spores directly on the gills of fish does not lead to infection. Other fish-infecting microsporidia have been transmitted by IP, IM, and waterborne (i.e. food, spore solution) exposure. The route of L.  peros, salmonae  development within the host after natural per os exposure is still unclear.  After injecting a sporoplasm into an epithelial cell of the alimentary canal L.  salmonae  may enter the lamina propria and gain access to the blood stream. Once in the blood it may reach its preferred site of development, the gills. Because L. salmonae  infects  endothelial cells it may also be found in other vascularized areas of the host. Some have suggested that fish-infecting microsporidia use macrophages or neutrophils as a transport mechanism. Whereas this may be possible, it has not been experimentally demonstrated. Macrophages ingest and destroy spores and IV injection of purified spores can infect fish. The latter suggests that L. salmonae  may reinfect the host when  spores are released from xenomas. Future studies should examine the route of L. salmonae  development within the host using in situ hybridization and the PCR probes  that have been developed. Some of this work is currently underway by GJ Sanchez at the Atlantic Veterinary College in Prince Edward Island, Canada. The importance of natural (peros,  salmonae L. salmonae  cohabitation) transmission routes used by L.  should not be overlooked. Fish-farmers often experience yearly epizootics of and this may be due to maintaining multiple fish year classes at one site.  Stocking of naive smolts next to infected market fish could result in infection of the smolts maintaining L. salmonae  at farm sites overtime. Fish may also be infected at the  hatchery, especially if the hatchery uses surface water with wild salmonids. Viability Under Various Laboratory Conditions. Viability was measured indirectly by 147  percent of spores excluding the dye methylene blue, and percent extruding a polar filament when placed in a hydrogen peroxide solution. Viability was also measured directly by infectivity of spores to fish using per os administration of spores. Methylene blue overestimated spore viability while extrusion rates underestimated spore viability when compared with infectivity of estimates from experimental infections. Spore viability decreased overtime and when spores were exposed to extreme conditions (i.e., heating, freezing). Spores were killed by rapid or slow freezing and use of 100 and 200 ppm iodophor disinfectant. Use of the iodophor on heavily-infected gill tissue reduced intensity of infections and delayed detection of them but did not completely kill all spores. Future work should examine what iodophor concentration is needed to kill all spores in heavily-infected gill homogenates. Pacific salmon farmers should continue to use iodophor to disinfect salmon eggs especially in handling of eggs from broodstock infected with L. salmonae.  Although vertical transmission has not been demonstrated, it  is a possibility because the parasite has been found in association with ovarian tissue. Loma  salmonae  spores stored at 4°C were viable for 95 d in fresh water and sea  water. The maximum viability of spores under those conditions should be determined and alternative methods of laboratory storage (e.g. cell culture) should be explored.  Host Susceptibility.  Loma  species of Oncorhynchus.  salmonae  is specific to salmonids and infects mainly  It did not infect Atlantic salmon or Arctic char, nor any  nonsalmonid tested, although it did infect brown trout. The parasite can also infect brook trout, and thus L. fontinalis  may be a synonym of L. salmonae.  This should be  investigated using sequencing of rDNA from both as differentiation of Loma spp. based on host, geography, and morphological criteria is of limited value in distinguishing species. Genetic and non-genetic factors interact to produce variations in susceptibility of 148  fish strains to a pathogen. Three strains of chinook were examined and the Northern Stream (NS) strain was consistently more susceptible and had a lower survivability when exposed to the parasite. Loma salmonae  may not be enzootic to the Yukon river  where this strain originates. Alternatively, other unknown genetic or non-genetic factors may play a role. Further study is necessary to quantify strain differences through breeding experiments and determine the factors that contribute to it. This information might be used by the aquaculture industry if they continue to select fish of the Southern Coastal (SC) strain, especially those that have been exposed to, and survived, infection by L.  salmonae.  Immunology. Fish respond humorally and cellularly to infections by microsporidia. Phagocytes such as macrophages may be especially important in innate or non-specific defense mechanisms of fish to microsporidia. This area is poorly understood and I investigated the ability of Atlantic salmon and chinook salmon macrophages to phagocytose L. salmonae  spores. Atlantic salmon exhibited a significantly higher  percent phagocytosis (PP) and phagocytic index (PI) than either Y or SC strains of chinook. This superior phagocytic ability may explain resistance of Atlantic salmon to L. salmonae.  Spores incubated in normal plasma were phagocytosed significantly more  than those incubated in heat inactivated plasma, no plasma, or EDTA inactivated plasma. Atlantic salmon may have a higher C titres, or other non-specific defense activity than chinooks. Alternatively, L. salmonae  may have evolved some mechanism  to avoid or suppress binding by macrophages of chinook. Prevention of phagosomelysosome fusion or stimulation of prostaglandin and/or leukotrien secretion has been noted in other fish-infecting microsporidia (Weidner & Sibley 1985; Laudan et al. 1989). Lectinophagocytosis of L. salmonae been suggested for a related Glugea  spores may also be important in chinook. This has sp. and fish-infecting microsporidia in general. 149  I did not determine a phagocyte to L. salmonae  spore ratio (R), and an optimal  ratio exists for each fish and pathogen species combination. I used a practical, approximate R value, based on purification time and ease at which phagocytosed spores could be enumerated. Future work should calculate an exact L. salmonae  R  value for the fish species being investigated. The R value might be used in assays requiring optimal phagocytic uptake of L. salmonae  spores. In addition to phagocytic  ability, other mechanisms may be involved in the response of a resistant species to a L. salmonae  challenge. No significant differences in PP or PI were found between the Y  and SC strain of chinook. Other mechanisms that must be investigated include respiratory burst activity of macrophages, and ultrastructural interactions between immune cells and spores. Finally, no information currently exists as to the mechanisms of resistance in Oncorhynchus  spp. to L.  salmonae.  Wild Salmonid and Nonsalmonid Hosts.  Loma  salmonae  was detected in wild  freshwater stocks of sockeye, coho, and chum salmon. The parasite was found in adult fish recently entering fresh water and smolts migrating seaward. Clinical signs were not present in smolts and the parasite was only detected using PCR. Loma  salmonae  may  contaminate the gravel so that fish become infected when they hatch. The infection might remain subclinical until fish mature at sea and begin their return migration. More extensive sampling of wild fish at different life history stages, and holding subclinically infected fish for greater than 1 y could provide data to support or refute this hypothesis. Loma  embiotocia,  was described in shiner perch based on host, geography,  morphology, and molecular (rDNA) characteristics. Previous Loma spp. descriptions show extreme overlap in morphological characters and L. embiotocia from L. salmonae  was differentiated  based mainly on differences in the ITS region of it's rDNA.  Transmission studies further support this and in light of the many Loma  spp. found 150  recently in marine nonsalmonids, it is recommended species be differentiated based on host, transmission studies, and molecular sequencing when possible. Currently, such work is ongoing by AMV Brown at the University of British Columbia, Vancouver, British Columbia, Canada.  151  BIBLIOGRAPHY  Abela M, Brinch-lversen J, Tanti J, Breton AL (1996) Occurrence of a new histozoic microsporidian (Protozoa, Microspora) in cultured gilt head sea bream Spams aurata L. 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Fish Pathol 25:141-146 Withler RE, Evelyn TPT (1990) Genetic variation in resistance to bacterial kidney disease within and between two strains of coho salmon from British Columbia. Trans Am Fish Soc 119:1003-1009 Wongtavatchai J, Conrad PA, Hedrick RP (1995a) In vitro characteristics of the microsporidian: Enterocytozoon salmonis. J Eukaryot Microbiol 42:401-405 Wongtavatchai J, Conrad PA, Hedrick RP (1995b) Effect of the microsporidian Enterocytozoon salmonis on the immune response of chinook salmon. Vet Immunol Immunopathol 48:367-374 Wyrick RB, Brownridge EA (1978) Growth of Chlamydia Immun 19:1054-1060  psittaciln  macrophages. Infect  Yokoyama H, Ogawa K, Wakabayashi H (1990) Chemotherapy with Fumagillin and toltrazuril against kidney enlargement disease of goldfish caused by the myxosporean Hoferellus carassii. Fish Pathol 25:157-163 Zar JH (1984) Biostatistical analysis, 2  nd  edn. Prentice Hall, New Jersey, p 185-243  Zhu X, Wittner M, Tanowitz HB, Cali A, Weiss LM (1994) Ribosomal RNA sequences of Enterocytozoon bieneusis, Septata intestinalis and Ameson michaelis: Phylogenetic construction and structural correspondence. J Eukarot Microbiol 41:204-209 Zhuo LS, Sajdak L, Phillips RB (1994) Minimal intraspecific variation in the sequence of the transcribed spacer regions of the ribosomal DNA of lake trout (Salvelinus namaycush). Genome 37:664-671  172  APPENDIX I Peer Reviewed Publications  Shaw RW, Kent ML (1999) Fish microsporidia. In: Wittner M, Weiss LM (eds) The microsporidia and microsporidiosis. ASM Press, Washington, DC, p 418-446 Shaw RW, Kent ML, Adamson ML (1998) Modes of transmission of Loma (Microsporidia). 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