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The salmon louse Lepeophtheirus salmonis (Caligidae) as a vector of Aeromonas salmonicida Novak, Colin William 2013

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    THE SALMON LOUSE LEPEOPHTHEIRUS SALMONIS (CALIGIDAE) AS                            A VECTOR OF AEROMONAS SALMONICIDA  by  Colin William Novak B.Sc., Vancouver Island University, 2009   A THESIS SUBMITTED IN PARTIAL FULLFILLMENT OF                                                THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE  in  THE FACULTY OF GRADUATE STUDIES  (Animal Science)   THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)  July 2013  ? Colin William Novak, 2013 ii  Abstract The sea louse, Lepeophtheirus salmonis, has been hypothesized to be a vector of fish pathogens and previous studies have isolated viral and bacterial pathogens from L. salmonis parasitizing farmed salmon. To examine the potential transmission of A. salmonicida by preadult and adult L. salmonis via parasitism of Atlantic salmon (Salmo salar), an in vivo bacteria-parasite challenge model was tested. Two pathogen challenge trials were performed, in which sea lice from donor (Aeromonas salmonicida-injected) fish were allocated among recipient fish for 14 days. Three hypotheses were examined: (i.) L. salmonis can acquire A. salmonicida from donor fish via parasitism; (ii.) A. salmonicida-exposed sea lice can transmit the pathogen to recipient Atlantic salmon via parasitism and (iii.) L. salmonis and A. salmonicida infections can cause synergistic effects on host fish. Sea lice acquired A. salmonicida externally (Trial 1 and 2, 100%) and internally (Trial 1, 100%) from parasitizing donor fish. Trial 1 (~44g fish) demonstrated a successful transfer of bacteria from lice to salmon (mucus, 100%; kidney, 77.3%), with a decrease (t = 5.29, df = 6, p = 0.00186) in mean fish condition factor and 59.1% cumulative fish mortality. Conversely, there was no evidence of bacteria transfer, no fish mortality and no decrease in mean fish condition factor in Trial 2 (~155g fish). In addition, histological examination revealed widespread inflammatory responses in small salmon (~46 g) infected with A. salmonicida and sea lice. Thus, preadult and adult L. salmonis can acquire and transmit A. salmonicida to recipient fish via parasitism under experimental conditions. However, the following conditions of pathogen, environment and host are required: (i.) a large inoculum of A. salmonicida (106 - 107 colony forming units (CFU) mL-1), (ii.) internal acquisition of bacteria by sea lice and, (iii.) young Atlantic salmon post-smolts (~44g) as hosts.   iii  Preface Chapter 2 has been submitted for publication. I am first author and I wrote most (~95%) of the manuscript. The section on sea lice in A. salmonicida water baths was originally drafted by D. E. Barker and is based on work conducted by D. L. Lewis, K. Verkaik and B. Collicutt at Vancouver Island University (VIU). I was responsible for the in vivo bacteria-parasite challenge models containing sea lice, A. salmonicida and Atlantic salmon, conducted fish and sea lice sampling, and performed daily water parameter tests. This research was performed under VIU animal use protocols 2010-05 TR (15/6/2010 ? 14/6/2011) and 2010-05 TR-1 (15/6/2011 ? 15/6/2012). Some aspects of chapter 2 have been published as a long abstract in the Aquaculture Association of Canada 2011 conference proceedings. Novak, C., Barker, D. and McKinley, S. 2011. First evidence of the salmon louse, Lepeophtheirus salmonis (Caligidae), as a vector of Aeromonas salmonicida. AAC Special Publication. 20:84-87. I conducted all testing and wrote the manuscript. The Appendices section is based on work conducted in the Centre of Shellfish Research building (VIU) and the Pacific Biological Station (PBS) in Nanaimo, BC. I conducted most of the testing. The skin plugs and histology performed in section ?B-Pilot Study? was conducted by L. M. Braden (Ph.D. candidate, University of Victoria), and was not used in her thesis.       iv  Table of Contents  Abstract ........................................................................................................................................ ii Preface ......................................................................................................................................... iii Table of Contents ....................................................................................................................... iv List of Tables .............................................................................................................................. vi List of Figures ............................................................................................................................ vii List of Abbreviations ............................................................................................................... viii Acknowledgements ..................................................................................................................... x Dedication ................................................................................................................................... xi 1 Introduction ..................................................................................................................... 1  1.1 Ectoparasites as Vectors ............................................................................................. 1  1.2 Sea Lice as Vectors ..................................................................................................... 3  1.3 Aeromonas salmonicida as a Model Pathogen ........................................................... 9  1.4 Objectives ................................................................................................................. 12 2 Transmission of Aeromonas salmonicida Via a Possible Vector: the Salmon                           Louse Lepeophtheirus salmonis (Caligidae) ................................................................ 14  2.1 Introduction ............................................................................................................... 14         2.2 Materials and Methods .............................................................................................. 17  2.2.1 Sea Lice Collection ................................................................................................ 17  2.2.2 Experimental Fish and Holding Conditions ........................................................... 18  2.2.3 Aeromonas salmonicida Preparation ..................................................................... 19   2.2.4 Bacteriology and A. salmonicida Confirmation from Lice and Fish ..................... 19  2.2.5 Sea lice and A. salmonicida Challenges ................................................................ 20  2.2.6 Data Analyses ........................................................................................................ 22  2.3 Results ....................................................................................................................... 24  2.3.1 Hypothesis 1........................................................................................................... 24  v  2.3.2 Hypothesis 2........................................................................................................... 25  2.3.3 Hypothesis 3........................................................................................................... 26  2.4 Discussion ................................................................................................................. 32  3 General Discussion ........................................................................................................ 38     3.1 Sea Lice Acquisition of A. salmonicida .................................................................... 38  3.2 Aeromonas salmonicida Transmission Via Sea Lice................................................ 41  3.3 Pilot Experiment ....................................................................................................... 46  3.4 Serendipitous Discoveries ......................................................................................... 48  3.5 Future Research ........................................................................................................ 49  3.6 Research Implications ............................................................................................... 51 Works Cited ............................................................................................................................... 54 Appendices ................................................................................................................................. 65     Appendix A: Supplemental Data .................................................................................... 65  Appendix B: Pilot Study ................................................................................................. 67  Appendix C: Serendipitous Discoveries ......................................................................... 70           vi  List of Tables  Table 2.1 Husbandry Conditions of Experimental Fish and Sea Lice Density .......................... 24 Table 2.2 Data Collected from Sea Lice Parasitizing Experimental Fish................................... 29 Table A.1 Mean Seawater Parameters in Tanks Containing Treatment Fish ............................. 66                    vii  List of Figures  Figure 1.1 Lepeophtheirus salmonis Life Cycle .................................................................... 4 Figure 2.1 Mean Daily Cumulative Percent Mortality of Salmo salar Parasitized                          with Aeromonas salmonicida-infected Sea Lice................................................. 30 Figure 2.2 Mean Fish Condition Factor of Reference and Recipient S. salar Before                      and After Lepeophtheirus salmonis Infections ................................................... 31 Figure A.1 Cumulative Daily Percent Mortalities of S. salar Ip-injected with                               A. salmonicida and Infected with L. salmonis .................................................... 65 Figure A.2 Lepeophtheirus salmonis Feeding on an Atlantic Salmon .................................. 65 Figure A.3 Mean Number of L. salmonis Feed Sites on Reference and Recipient S. salar . 66 Figure B.1 Histological Skin Samples of S. salar Parasitized by L. salmonis...................... 68 Figure B.2 Histological Skin Samples of S. salar Parasitized by L. salmonis and           Infected with A. salmonicida .............................................................................. 69 Figure C.1 Kudoa thyrsites Infections in Three Reference Fish from Trial 2 ...................... 70 Figure C.2 Antithamnion sp. Growing on the Carapace of L. salmonis ............................... 71        viii  List of Abbreviations ASW ? autoclaved sea water. CBB ? Coomassie brilliant blue agar, a differential agar media for Aeromonas salmonicida. CFU ? colony forming units, a measurement of bacterial colonies growing on an agar plate. DO ? dissolved oxygen, a measurement of oxygen in water (mg L-1). dpi ? days post injection, measurement of time in days after the initial injection of A. salmonicida in fish. FS ? sea lice feeding sites observable on host fish. I ? mean intensity, mean number of L. salmonis attached to infected fish. IHNV ? Infectious Haematopoietic Necrosis Virus. Ip ? intraperitoneal, an injection of a substance into the peritoneum (body cavity). IPNV ? Infectious Pancreatic Necrosis Virus. K ? Fulton?s condition factor [K = (weight / fork length3) ? 100], which describes the relative health ?condition? of each individual fish. Weight in grams, length in cm. LPS ? Lipopolysaccharide, major component of outer membrane of Gram-negative bacteria. P ? prevalence, percentage of fish infected with lice or A. salmonicida. R ? range of infection, the minimum to maximum number of lice per fish. R2 ? goodness of fit. ix  r ? correlation coefficient. RP ? recovery percent, number of recovered lice divided by original number of lice used in infection. T ? temperature (?C). TMS ? Tricaine Methanesulfonate.            x  Acknowledgements This work was funded by a Natural Sciences and Engineering Research Council (NSERC) of Canada Strategic Projects Grant (STPGP 372605-08) awarded to D. E. Barker, K. Garver and S. R. M. Jones in collaboration with the department of Fisheries and Oceans Canada and Marine Harvest Canada (MHC). Brad Boyce from MHC was instrumental in louse collection and both VIU and PBS provided facilities. The technical assistance of Danielle Lewis (UBC, M.Sc. candidate), Laura Braden (UVic, Ph. D. candidate), Katie Verkaik (VIU, B.Sc.) and Brenna Collicutt (VIU, B.Sc.) is greatly acknowledged. Moreover, thank you to D. E. Barker, C. J. Brauner, M. A. G. von Keyserlingk and R. S. McKinley for providing valuable comments.          xi  Dedication I wish to dedicate this thesis to my beautiful wife (Joanne Novak), who has been there beside me through this whole process and my parents (Dan and Wendy Vircik), who have always supported me in all my adventures.                1 Introduction 1.1 Ectoparasites as Vectors Vectors, in biological terms, are defined as organisms that transmit parasites and/or pathogens from one host to another (Pechenik, 2005; Roberts and Janovy, 2005) and are often categorized as biological or mechanical. Biological vectors transmit a pathogen which undergoes development inside the vector (Gullan and Cranston, 2005). The pathogen needs its biological vector to persist and complete its life cycle. Well known examples include tsetse flies (Glossina spp.) that transmit Trypanosoma brucei (African sleeping sickness), Anopheles mosquitoes that transmit Plasmodium spp. (malaria) and Ctenocephalides fleas that transmit Dipylidium caninum tapeworms. The protozoan T. brucei migrates to the salivary glands of the tsetse fly where it generates metacyclic forms that are infectious for mammals (Roditi and Lehane, 2008). Inside the Anopheles mosquito midgut, Plasmodium undergoes three transformations leading to the sporozoite stage, which migrates to the salivary glands ready for transmission to a new host (Ramasamy et al., 1997). Dipylidium caninum eggs ingested by fleas hatch and develop into oncosphere larvae that become cysticercoid larvae, which are infective to mammal hosts (Roberts and Janovy, 2005). By contrast, mechanical vectors (transport hosts) transmit pathogens that do not undergo development while associated with the vector (Gullan and Cranston, 2005). The pathogen does not need its mechanical vector for survival nor is it essential to its life cycle. However, the mechanical vector does benefit the pathogen by increasing its dispersal and ability to infect new hosts. Examples of mechanical vectors include Musca houseflies that transiently carry the bacteria Shigella spp. (causative agent of dysentery) on their surfaces or within their gastrointestinal tracts (Levine and Levine, 1991). Ixodes ticks transmit the bacteria Borrelia 2  burgdorferi, causative agent of Lyme disease to humans (Soares et al., 2006). The above examples are from well documented terrestrial ectoparasitic arthropods that transmit pathogens.  Several fish ectoparasites have been hypothesized to be vectors for pathogens of fish. Cusack and Cone (1986) reviewed this topic and noted the majority of the potential parasite vectors were monoxenous (one-host, direct life cycle) tissue feeding species. They describe the role of the vector as the ability to breach the host epidermis and transfer directly from fish to fish. The freshwater fish louse Argulus foliaceus L. and the leech Piscicola geometra L. were experimentally observed to act as mechanical vectors of Rhabdovirus carpio (spring viraemia of carp virus) in carp (Ahne, 1985). Becker and Katz (1965) discovered Rhynchobdellid leeches were able to transmit the haemoflagellate Cryptobia salmositica to coho salmon (Oncorhynchus kisutch) and torrent sculpins (Cottus rhotheus). Overstreet et al. (2009) experimentally discovered the Taura syndrome virus can replicate for at least two weeks and establish an infective dose in three parasitic crustacean vectors: the copepod Ergasilus manicatus on the gill filaments of the Gulf killifish (Fundulus grandis); the acorn barnacle (Chelonibia patula) on the carapace of the blue crab (Callinectes sapidus) and the gooseneck barnacle (Octolasmis muelleri) also on the gills of C. sapidus. Infectious haematopoietic necrosis virus (IHNV) was isolated from both a leech Piscicola salmositica and a copepod Salmincola sp. (Mulcahy et al., 1990), while viral hemorrhagic septicaemia virus (VHSV) was detected in a leech Myzobdella lugubris (Faisal and Schulz, 2009) and from an amphipod zooplankton Diporeia sp. (Faisal and Winters, 2011). Aeromonas salmonicida has been isolated from sea lamprey (Petromyzon marinus) (El Morabit et al., 2004) and marine plankton (Nese and Enger, 1993) and bacterial microcolonies were consistently observed on the surface of Gyrodactylus avalonia (Cusack and Cone, 1985). 3  One limitation of the majority of these studies is that the aquatic ectoparasites were not experimentally tested as vectors.  1.2 Sea Lice as Vectors Two species of sea lice (Copepoda: Caligidae), Lepeophtheirus salmonis Kr?yer and Caligus clemensi Parker and Margolis, are commonly reported parasitizing farmed and wild salmon off the coast of BC (Beamish et al., 2005, 2006, 2007, 2009; Saksida et al., 2007a, b). Caligus clemensi was first recorded from pink (Oncorhynchus gorbuscha), coho and chum (O. keta) salmon (Parker and Margolis, 1964) and is regarded as a generalist species found on many teleosts from Pacific Canadian waters (Gottesfeld et al., 2009). Parker and Margolis (1964) reported that C. clemensi showed no evidence of host specificity and suggested the parasite will infect any fish species inhabiting inshore coastal waters. Caligus clemensi is the most common caligid on the Pacific northeast coast, but it is L. salmonis that is of special interest because it is the most abundant parasite on Atlantic salmon farms off the coast of BC (Pike and Wadsworth, 1999; Saksida et al., 2007a, b). The first problems of L. salmonis infections on Atlantic salmon farms occurred in Norway during the 1960s (Pike and Wadsworth, 1999). Since then, similar problems have been observed in Scotland, Ireland and along the east and west coast of North America (Pike and Wadsworth, 1999). Most studies report L. salmonis to be more numerous than C. clemensi on all major species of juvenile and adult pacific salmon off the west coast of BC (Beamish et al., 2005) and in the central subarctic Pacific Ocean (Nagasawa et al., 1993, 1998). Lepeophtheirus salmonis is regarded as being more host specific than C. clemensi, having been reported from 12 species most commonly within the genera Salmo (Atlantic salmon and trout), Salvelinus (trout and charr) and Oncorhynchus (Pacific salmon) (Pike and Wadsworth, 1999). However, the copepod has also been reported from white sturgeon (Acipenser transmontanus), 4  sand lance (Ammodytes hexapterus) (Kabata, 1973), saithe (Pollachius virens) (Bruno and Stone, 1990) and two species of cyprinids (Nagasawa, 2004). Recently, the three-spine stickleback, Gasterosteus aculeatus L., has also been reported as an alternative and/or temporary host for both L. salmonis and C. clemensi (Jones et al., 2006a, b; Jones and Prosperi-Porta, 2011). Lepeophtheirus salmonis has a circumpolar distribution in the Northern Hemisphere where it thrives in temperate to subarctic areas. Recent research has revealed the Atlantic and Pacific populations of L. salmonis are distinct (Tjensvoll et al., 2006; Yazawa et al., 2008). There is a reduced genetic diversity within the Pacific form which suggests L. salmonis was introduced to the Pacific Ocean from the Atlantic Ocean via the opening of the Bering Strait, approximately 5 million years ago (Yazawa et al., 2008). Since then, the two forms of L. salmonis have coevolved independently.   Figure 1.1 Lepeophtheirus salmonis Life Cycle.      http://www.upei.ca/~anatphys/Sea_Lice/licecycl.htm 5  The life cycle of L. salmonis contains 10 stages with both free-living and parasitic forms (Fig. 1.1). The first three life stages (nauplius I, II and copepodid) are planktonic and non-feeding, relying on their energy reserves within their yolk sac (Boxaspen, 2006). The nauplius I (mean duration of 3.5 h at 10oC) and nauplius II (duration: 56.9 h at 10oC) stages are intermittent swimmers that are photopositive (Johannessen, 1977; Johnson and Albright, 1991; Pike et al., 1993). The energy supply in copepodids (7800 cal g-1 dry weight) rapidly declines within seven days (Tucker et al., 2000), giving the actively swimming copepodid a narrow window to find a host, settle and continue development (Johnson and Albright, 1991). Once the copepodid has settled on a host, the chalimus I stage develops and attaches (via a specialized short, frontal filament) to the host (Pike and Wadsworth, 1999). All four successive sessile chalimus stages (I ? IV) feed exclusively on the host mucus and epidermis around their restricted point of attachment (Brandal et al., 1976; Pike and Wadsworth, 1999). Sexual maturation begins at the two motile preadult stages (I-II), ultimately leading to the adult form (Boxaspen, 2006). The preadult and adult stages are motile browsers that can move along the epidermis of their host and transfer among hosts (Pike and Wadsworth, 1999). The complete life cycle has been observed to take 40 days for males and 52 days for females under laboratory conditions at 10oC (Johnson and Albright, 1991). The optimal temperature and salinity ranges for L. salmonis are 10-15oC and 25-30? with lower development times correlated to higher temperatures and salinities (Johnson and Albright, 1991). However, a salinity of 30? is required for nauplii to develop to copepodids (Johnson and Albright, 1991).  Preadult and adult stages of L. salmonis can transfer between host fish. Ritchie (1997) observed adult males and preadults of both sexes to be more motile among S. salar than adult females. He observed that the majority of adult male lice changed hosts within 24 h, while 6  preadult females remained on initial hosts for longer periods. Furthermore, Connors et al. (2008) demonstrated (in 70% of their trials) that sea lice can escape predation by swimming or moving directly onto the predator. This trophic transmission was strongly male-biased, which may be a result of sea lice being a polygynous species (Connors et al., 2008). Male lice fitness is dependent on the number of successful matings and female lice fitness is dependent on energy and nutrient reserves for egg production (Connors et al., 2008). Thus, male lice move along hosts to increase mate encounters and female lice remain on hosts to obtain resources (Connors et al., 2008). The distribution of sea lice on a host differs depending on host age, sea lice stage/sex, competition for mates, and avoidance interactions (Pike and Wadsworth, 1999). It is the active, motile behaviour of preadult and adult L. salmonis that makes them suitable vector candidates. Host susceptibility to L. salmonis infections is based on several interacting factors including stress and nutrition, host size, immunocompetence and the genetically predetermined susceptibility (MacKinnon, 1998). Post-smolt salmonids are most vulnerable to L. salmonis infection (Finstad et al., 2000; Jones et al., 2008). Although, pink salmon show a functional resistance at 0.7 g coinciding with changes in immune responses within the epidermis and dermis (Jones et al., 2008). There is also a difference in host susceptibility among salmonid species. Experimentally, Atlantic and chum salmon were more susceptible to infection by L. salmonis than the more resistant coho, pink and chinook salmon (Johnson and Albright, 1992; Jones et al., 2007). This variability among host species is associated with differences in the inflammatory response elicited by L. salmonis attachment and feeding (Johnson and Albright, 1992; Fast et al., 2002; Jones et al., 2007). Resistant species exhibit epidermal hyperplasia and associated inflammation of underlying tissues to aid parasite rejection (Johnson and Albright, 1992). Seasonal overlapping distributions of wild Pacific salmon (Oncorhynchus spp.) co-existing with 7  farmed Atlantic salmon (S. salar) off the coast of BC, Canada provides a unique host-parasite system to study because of the possibility of cross-infestation of sea lice and any implications for vector transmission.  Lepeophtheirus salmonis feed on mucus, epidermis and blood of host fish using a muscular apparatus at the tip of the labium called the strigil (Kabata, 1974). The strigil contains about 100 fine, sharp teeth that are used in a sawing movement (Kabata, 1974). The resulting accumulation of tissue debris is picked up by mandibular teeth, acting as conveyers, moving fish tissue into the buccal cavity (Kabata, 1974). This feeding behaviour inflicts mechanical damage to host fish which can lead to several host problems. The severity of these problems is determined by a number of factors: dynamic interactions between host and parasite, parasite stage, number of lice present, host age and species, genetic strain, physiological condition and position in the population hierarchy (Pike and Wadsworth, 1999). Lepeophtheirus salmonis are not life threatening to fish unless the infection levels increase beyond the host?s ability to compensate (Pike and Wadsworth, 1999). Infections can lead to host stress, open sores, lesions, and in extreme examples, death via osmoregulatory failure or secondary infection (Pickering and Pottinger, 1989; Bowers et al., 2000; Boxaspen, 2006). The development of chronic stress may result in immunosuppression, and thereby increase host susceptibility to secondary infections (Mustafa et al., 2000; Bowers et al., 2000). Also, epidermal erosion can lead to the loss of host body fluids including blood, lymph, protein and electrolytes, and the release of cortisol and its immunosuppressive effects (Grimnes and Jakobsen, 1996; Pike and Wadsworth, 1999). Plasma chloride levels have been reported to increase, while total protein, haematocrit, and albumin decrease in Atlantic salmon with a high louse infection (>30 lice fish-1, ~40 g) (Grimnes and Jakobsen, 1996). Moreover, cortisol and glucose levels increase (Bowers et al., 2000), while 8  haematocrit, sodium and cholesterol levels decrease (Dawson et al., 1999) in Atlantic salmon after a 21 day artificial infection (>100 lice fish-1). Histological changes have also been observed in the skin and gill epithelia of Atlantic salmon infected with L. salmonis, including: increased apoptosis, necrosis, leukocyte infiltration, edema, hyperplasia, sloughing of cells and cellular inflammation (J?nsd?ttir et al., 1992; Nolan et al., 1999). Lepeophtheirus salmonis infections may also alter the hosts? behaviour. Grimnes and Jakobsen (1996) reported a form of fish irritability with increased leaping and rolling (fish quietly breaking the water surface) by Atlantic salmon post smolts experimentally infected with copepodids. Inappetence may also be common during moderately severe infections (Dawson, 1997). Furthermore, biochemical changes have been observed in the mucus layer at the site of the host-parasite interaction, with an increase in protease activity over time (Ross et al., 2000).  Previous studies have documented the isolation of bacteria (Nylund et al., 1991) and bacterial pathogens including A. salmonicida (Nese and Enger, 1993), Tenacibaculum maritimum, Pseudomonas fluorescens and Vibrio spp. (Barker et al., 2009) from the outer surface and midgut of the sea louse Lepeophtheirus salmonis. In addition, A. salmonicida (Nese and Enger, 1993), salmon alphavirus SAV3 RNA (Petterson et al., 2009), and infectious salmon anemia virus (Nylund et al., 1991, 1993) have been isolated from L. salmonis attached to clinically diseased fish. Until recently, studies such as these hypothesized sea lice could be suitable vectors of bacterial and viral pathogens. Jakob et al. (2011) were the first to document that adult L. salmonis can act as mechanical vectors of IHNV and transmit it to unexposed Atlantic salmon (Salmo salar) through parasitism under experimental conditions. The sea lice were able to acquire IHNV through water bath exposure as well as from host parasitism on IHNV-positive Atlantic salmon, and the sea lice remained virus-positive for 12 - 24 h post 9  exposure. Cusack and Cone (1986) hypothesized that vector movement between hosts may contribute to the dissemination of pathogens, especially obligate microparasites (IPNV, A. salmonicida and Renibacterium salmoninarum) that do not survive for extended periods in water. However, the question remains whether these pathogens are dependent on such vectors for transmission. 1.3 Aeromonas salmonicida as a Model Pathogen Aeromonas salmonicida, one of the oldest described fish pathogens, was first isolated and reported (Emmerich and Weibel, 1894, as cited in Austin and Austin, 2007) from diseased brook trout (Salvelinus fontinalis). Eight years later, the first report of A. salmonicida in North America (Michigan, USA) was documented (Marsh, 1902, as cited in Austin and Austin, 2007). Since then, A. salmonicida has been found to have a worldwide distribution, infecting over 70 fish species (Austin and Austin, 2007; Bernoth et al., 1997). Early recognition of this pathogen and its commercial importance has resulted in it being one of the most studied bacterial pathogens of fish (Brown and Johnson, 2008). It is a non-motile, catalase and oxidase positive, fermentative, small (0.8-1.0 x 1.5-2.0 ?m) Gram-negative rod (bacilli) that does not grow at 37oC (Roberts, 1978; Austin and Austin, 2007). There are two strains of A. salmonicida - typical and atypical. The typical strain is the classic description of A. salmonicida, while atypical strains deviate by a number of biochemical, physiological and genetic properties and tend to occur more frequently among non-salmonids (Austin and Austin, 2007). The typical strain of A. salmonicida contains an additional layer (A-layer) on its lipopolysaccharide (LPS) layer, initially described by Udey and Fryer (1978). The A-layer protein contains a high proportion of hydrophobic and uncharged amino acids (Trust et al., 1983) covering most of the cell surface and consequently allowing 10  penetration only by O polysaccharide chains of the LPS layer (Ishiguro et al., 1981; Kay et al., 1981). Aeromonas salmonicida causes the disease furunculosis, notably known for forming necrotic lesions in the skin and muscles of fish hosts in its chronic form (Austin and Austin, 2007; Bernoth et al., 1997). The chronic form, more common in older fish, is characterized by lethargy, slight exophthalmia, haemorrhagic fins, bloody discharge from the nares and vent, multiple haemorrhages in the muscle and other tissues, swelling in the spleen and kidney necrosis (Austin and Austin, 2007). The chronic form causes low rates of mortality with fish often surviving, although scar tissue is often present near the furuncle (Austin and Austin, 2007). The acute form is more common, particularly in growing fish and adults (Austin and Austin, 2007). Fish with acute infections exhibit general septicaemia, melanosis, inappetence, lethargy, an enlarged spleen, small haemorrhages at the base of the fins and fish usually die in 2 to 3 days (Austin and Austin, 2007). More often the disease begins suddenly with few, if any, external signs (Austin and Austin, 2007). Lastly, the peracute form is confined to fingerling fish, which exhibit only slight external symptoms (i.e., mild exophthalmia) due to quick death and limited immune response (McCarthy and Roberts, 1980). The site of entry of A. salmonicida in the fish remains uncertain. Possible entry points may be through the gills, lateral line, mouth, anus, and/or surface injury (Bernoth et al., 1997). It is known that infections can be obtained from contact with infected fish or contaminated water, fish farm materials and transovarian transmission (Austin and Austin, 2007). Some species of fish that recover from a furunculosis epizootic are capable of acting as carriers, harbouring low levels of the virulent pathogen in their tissues and kidney (McCarthy, 1977; Bernoth et al., 1997), but they have the potential for transmission (Bernoth et al., 1997). Nordmo et al. (1998) described the best mimic to a natural 11  furunculosis infection to be via the cohabitation method after i.p. injection. This method requires intraperitoneally injecting fish with A. salmonicida, termed the ?cohabitant? fish, and placing them with na?ve fish to induce infections. Aeromonas salmonicida is categorized as an obligate pathogen but can survive for limited periods in freshwater, seawater and in sediment beneath net pens containing fish (Enger and Thorsen, 1992; Bernoth et al., 1997). The pathogen has adapted to a copiotrophic life style that requires high concentrations of nutrients for active growth (Bernoth et al., 1997). There are contradictory results of survival times in different aquatic environments. McCarthy (1980) reported the pathogen to survive between 24 h and 8 days in unsterilized seawater, while Lund (1967) described survival time to greatly increase (up to 24 d) in sterilized seawater due to the absence of competing organisms. Rose et al. (1990) examined survival times of bacteria grown both in vivo and in vitro in different types of microcosms (glass or dialysis bags) and in sterile and non-sterile sea water. They found survival of A. salmonicida to be of short duration (< 10 days) in all treatments. In addition, A. salmonicida abundances were found 1-2 orders of magnitude higher at the air-water interface than in subsurface water because of its hydrophobic properties (Enger et al., 1992). These studies concluded that the concentration of inoculum and temperature are important factors affecting the outcome of A. salmonicida survival studies. Furthermore, McCarthy (1977) observed A. salmonicida cells in water to increase once host fish were dead. This discovery along with Enger et al. (1992) revealed that diseased and especially dead fish were the main source of water-borne A. salmonicida. Thus, the spread of A. salmonicida cells depends on its rate of shedding from infected fish and prevailing hydrographic conditions (Rose et al., 1990). 12  Historically, salmonids were regarded as the primary host of A. salmonicida (Mackie et al., 1930). Currently, many freshwater and marine fish species have become recognised as susceptible. Susceptibility to A. salmonicida infections has been observed among different salmonid hosts, stages and strains. Cipriano and Heartwell III (1986) reported furunculosis caused the highest mortalities among brown trout (S. trutta), followed closely by brook trout, but few mortalities were reported for rainbow trout (S. gairdneri). Similarly, cumulative mortality rates of Atlantic (Nordmo et al., 1998; Nordmo & Ramstad, 1999), chinook, coho, and chum salmon (Beacham & Evelyn, 1992; Ogut & Reno, 2005) in freshwater cohabitation studies have all shown variability. Rainbow trout are the most resistant, Pacific salmon have moderate resistance and Atlantic salmon, brook trout and brown trout are very susceptible (Bernoth et al., 1997). Pickering and Duston (1983) confirmed that chronic elevation of plasma cortisol levels (resulting in immunosuppression) increases furunculosis infections, leading to mortality in brown trout.  1.4 Objectives The motility and blood-feeding behaviour of the ectoparasitic copepod L. salmonis in the presence of an obligate pathogen (A. salmonicida) and susceptible host (S. salar) make an ideal ?model? system to study. Although previous studies (Nylund et al., 1991; Nese and Enger, 1993; Barker et al., 2009) had hypothesized the vector potential of L. salmonis, it was not until recently that it was experimentally tested and the copepod was shown to act as a mechanical vector of IHNV, transmitting the pathogen to Atlantic salmon (Jakob et al., 2011). The current study examined the potential role of L. salmonis acting as a vector of the pathogenic bacterium A. salmonicida among Atlantic salmon. In particular, preadult and adult L. salmonis transmission of A. salmonicida from infected fish to uninfected fish via parasitism using an in vivo bacteria-13  parasite challenge model was tested. This was accomplished by performing three experimental challenges specifically addressing the following objectives:  (i) Test if L. salmonis can acquire A. salmonicida from i.p.-injected fish via parasitism;  (ii) Examine the transmission ability of A. salmonicida-exposed sea lice to uninfected fish via parasitism;  (iii) Test any synergistic effects of parasite and pathogen on the salmon host.           14  2 Transmission of Aeromonas salmonicida Via a Possible Vector: the Salmon Louse Lepeophtheirus salmonis (Caligidae) 2.1 Introduction  Vectors are organisms that transmit parasites and/or pathogens from one host to another (Pechenik 2005). Mechanical vectors transmit pathogens that do not undergo development while inside the vector; whereas, a biological vector is one in which the pathogen does exhibit development (Gullan and Cranston 2005). Either transmission method increases the pathogen?s ability to infect a new host. There are many well documented terrestrial ectoparasitic arthropods that are important vectors of pathogens. Ixodes ticks transmit the bacteria Borrelia burgdorferi (Lyme disease), Anopheles mosquitoes transmit Plasmodium (malaria) and Ctenocephalides fleas transmit Dipylidium caninum tapeworms. Similarly, fish ectoparasites, especially monoxenous tissue feeding species, have been hypothesized to be vectors for pathogens (Cusack and Cone 1986). To act as vectors, aquatic ectoparasites should be able to breach the host epidermis and transfer a pathogen from one fish to another (Cusack and Cone 1986). Rhynchobdellid leeches can transmit the haemoflagellate Cryptobia salmositica to coho salmon (Oncorhynchus kisutch) and torrent sculpins (Cottus rhotheus) (Becker and Katz 1965). Similarly, the freshwater fish louse Argulus foliaceus L. and the leech Piscicola geometra L. can act as mechanical vectors of Rhabdovirus carpio (spring viraemia of carp virus) in carp under experimental conditions (Ahne 1985). A variety of aquatic pathogens including Aeromonas salmonicida (Nese and Enger 1993), salmon alphavirus (SAV, Petterson et al. 2009), infectious salmon anemia virus (ISAV, Nylund et al. 1991, 1993), Tenacibaculum maritimum, Pseudomonas fluorescens and Vibrio spp. (Barker et al. 2009) have been isolated from the outer surface and midgut of the ectoparasitic arthropod sea louse, Lepeophtheirus salmonis, which suggests sea lice may act as vectors of aquatic pathogens. Cusack and Cone (1986) hypothesized 15  that the dissemination of obligate microparasites (A. salmonicida, Renibacterium salmoninarum and infectious pancreatic necrosis virus) that do not survive for extended periods in water may benefit from vector movement between hosts. To date, one study by Jakob et al. (2011) provides experimental evidence that adult L. salmonis can act as vectors of infectious haematopoietic necrosis virus (IHNV) and transmit it to na?ve Atlantic salmon (Salmo salar L.) through parasitism. IHNV was acquired by sea lice through pathogen concentrated water bath exposure or host parasitism on IHNV-positive Atlantic salmon (Jakob et al. 2011). However, it was concluded that the sea lice were mechanical vectors because the virus did not replicate while associated with the lice and the lice were virus-positive for only 12?24 h post exposure (Jakob et al. 2011).  Two species of sea lice, Lepeophtheirus salmonis Kr?yer and Caligus clemensi Parker and Margolis, are commonly reported parasitizing farmed Atlantic and wild Pacific salmon (Oncorhynchus spp.) off the coast of BC (Beamish et al. 2005, 2006, 2007, 2009, Saksida et al. 2007a, b). Lepeophtheirus salmonis is of special interest because it is the most abundant copepod parasite of farmed Atlantic salmon (Pike and Wadsworth 1999, Saksida et al. 2007a, b) and wild Pacific salmon (Oncorhynchus spp.) off BC (Beamish et al. 2005) and in the central subarctic Pacific Ocean (Nagasawa et al. 1993, 1998). The life cycle of L. salmonis contains 10 stages with free-living (two nauplii and one infective copepodid) and parasitic (four chalimus, two preadult and one adult) forms (Kabata 1973, Pike and Wadsworth 1999). The preadult and adult stages are motile browsers that can move along the epidermis of their host and transfer among hosts (Pike and Wadsworth 1999). Lepeophtheirus salmonis feed on the mucus, epidermis and blood of host fish using a strigil, which contains 100 sharp teeth used in a sawing movement (Kabata 1974). This feeding behaviour inflicts mechanical damage to host fish and can result in 16  many problems including stress, open sores, lesions and sometimes death via osmoregulatory failure or secondary infection (Pickering and Pottinger 1989, Bowers et al. 2000, Boxaspen 2006). The feeding behaviour, ability to harbor aquatic pathogens and motility of preadult and adult sea lice make L. salmonis excellent candidates as vectors.  Aeromonas salmonicida was first isolated and described from diseased brook trout (Salvelinus fontinalis) in German hatcheries and has since been reported to have a worldwide distribution, being isolated from over 70 species of fish (Bernoth et al. 1997). Due to its commercial importance, this Gram-negative bacterium is arguably one of the most studied bacterial pathogens of fish (Brown and Johnson 2008). Once a host is infected, A. salmonicida causes furunculosis, which in its chronic form is notably described as forming necrotic lesions (furuncles) in the skin and muscles of host fish (Austin and Austin 2007, Bernoth et al. 1997). The chronic form is more common in older fish and causes low rates of mortality, but the more frequently occurring acute form occurs in growing fish, resulting in sudden death (Austin and Austin 2007). The site of entry remains uncertain, but it is known that infections can occur from contact with infected fish or contaminated water, fish farm materials and transovarian transmission (Austin and Austin 2007). Atlantic salmon, brook trout (Salvelinus fontinalis) and brown trout (Salmo trutta) are the most susceptible species to furunculosis infections (Bernoth et al. 1997). Similarly, Atlantic salmon are also more susceptible to L. salmonis infections than other species of salmonids (Johnson and Albright 1992, Dawson et al. 1998).  The feeding behaviour of the ectoparasitic sea louse Lepeophtheirus salmonis in the presence of an obligate pathogen (Aeromonas salmonicida) and susceptible host (Salmo salar) make an ideal model system to study. Many studies have hypothesized sea lice to act as vectors, but only one study (Jakob et al. 2011) has demonstrated L. salmonis acting as a mechanical 17  vector of IHNV in Atlantic salmon. The present investigation further examines this hypothesis testing transmission of the bacterium A. salmonicida among Atlantic salmon. Specifically, the transmission of A. salmonicida by preadult and adult L. salmonis via parasitism from infected fish to na?ve fish using an in vivo bacteria-parasite challenge model was tested.  This was accomplished by performing experimental challenges that addressed four hypotheses: i.) L. salmonis can acquire A. salmonicida from water bath exposure; ii.) L. salmonis can acquire A. salmonicida from intraperitoneal (ip)?injected (donor) fish via parasitism; iii.) L. salmonis exposed to A. salmonicida can transmit the pathogen to Atlantic salmon via parasitism; and iv.) L. salmonis and A. salmonicida infections can cause synergistic effects on host fish. 2.2 Materials and Methods 2.2.1 Sea Lice Collection Motile male and female stages (preadult and adult) of Lepeophtheirus salmonis were obtained in various collections (Jan. 2010 ? Sept. 2011) from farmed Atlantic salmon (Marine Harvest Canada) during harvesting in BC, Canada. The farm sites were located in zones 3-2 and 3-3 of the BC Ministry of Agriculture (BCMA) fish health surveillance zones (BCMAL 2008). Sea lice (n = 200-450 per collection) were gently removed (using forceps) from the skin of fish, placed in 10?C aerated buckets containing autoclaved seawater (ASW) and kept chilled (8-10?C) in a cooling box for transportation. Sea lice were transported to Vancouver Island University (VIU, Nanaimo, BC), stored at 10?C and used within 24 h. Only lice exhibiting active swimming and attachment behaviour were used. A portion (5-15%) of each lice collection was sampled (described below) as a pre-screen to determine if A. salmonicida was present from previous exposure.  18  2.2.2 Experimental Fish and Holding Conditions Atlantic salmon (Salmo salar L., n = 750-800, mean wt = 15.9 g) were obtained (31/08/2010) from Freshwater Farms Hatchery (Marine Harvest Canada) in Duncan, BC, Canada. Fish were transported via an aerated 1000 L plastic tank (Dissolved oxygen (DO) = 11.7 mg L-1; Temperature (T) = 13.5?C; pH = 7.9), to the Pacific Biological Station (PBS), Nanaimo, BC. Fish were held in two ambient temperature pathogen-free freshwater tanks (Volume = 1.9 m3; T = 12-13?C) and subsequently smolted by increasing the seawater:freshwater ratio by 25% every 4 to 5 days. Prior to the challenge trial dates (14/3/2011, 6/9/2011), fish were transported via an aerated 1000 L plastic tank (Trial 1, DO = 15 mg L-1; T = 10.5?C; Trial 2, DO = 15 mg L-1; T = 12?C) to temperature-controlled, recirculated, pathogen-free seawater at VIU. Recirculated seawater was treated via UV sterilizers (31,820 ?W x s cm-2). Temperature (Oxyguard?), pH (Hanna? pH meter), dissolved oxygen (Oxyguard?) and salinity (refractometer) were monitored daily during the trials. Fish used in Trial 1 (n = 78, mean wt = 44.5 g) and Trial 2 (n = 78, mean wt = 155.2 g) were acclimated 14 days prior to each challenge. Photoperiod was kept at 12-h artificial light and 12-h dark and fish were fed daily, at 1.5% of their body weight, a pelleted (Trial 1, 2 mm; Trial 2, 6 mm) diet (BioOregon?). Feed was withheld for 24 h prior to all treatments. Atlantic salmon were anaesthetized via aerated immersion baths (3 mg L-1 AquacalmTM) for handling and were then carefully transferred (via dip nets) to 20 L aerated ?low anaesthetic? baths (1 mg L-1 AquacalmTM) to maintain light sedation while being exposed to sea lice. Sea lice were individually rinsed with sterile physiological saline (1% NaCl), carefully manipulated with sterile forceps and placed in the vicinity (~5 cm) of fish. Small mesh (1.5 mm) was placed around the outflow of each tank to prevent loss of sea lice. Moribund and dead fish 19  were carefully removed from tanks, examined for sea lice, and aseptically transferred to the laboratory for examination.  2.2.3 Aeromonas salmonicida Preparation Aeromonas salmonicida cultures (virulent strains 2005-120 for baths and 2011-247 for challenges) were obtained from the Culture Collection, PBS Fish Health Unit, Nanaimo, BC. The bacteria were first cultured on Difco? Tryptic Soy Agar (TSA) then colonies were inoculated in sterile saline, vortexed and transferred into 2 ml Eppendorf tubes. After centrifugation (10 s at 6600 rpm), the supernatant was removed, and the tubes were filled with a trypticase soy broth containing 15% glycerol. Cultures were stored at -80?C until needed. Prior to use, frozen samples were thawed (2-4 h) at room temperature and plated on TSA for 48-72 h. Pure colonies of A. salmonicida were suspended in sterile physiological saline (1% NaCl) at 22?C, adjusted to a 107 concentration using McFarland Turbidity Standards (Whitman 2004) and a dilution series (101 - 109 CFU mL-1) was plated for enumeration and dilution verification.  2.2.4 Bacteriology and Aeromonas salmonicida Confirmation from Lice and Fish Atlantic salmon and attached L. salmonis were aseptically sampled within 24 h of death. Prior to fish dissections, measurements of fish wet weight (0.1 g) and fork length (0.1 cm) were recorded, skin mucus swabs were taken along the ventral and dorsal surfaces using sterile cotton-tipped applicators and any abnormalities were noted. Attached sea lice were aseptically (using sterile forceps) removed and individually placed into 2.0 mL microcentrifuge tubes containing sterile physiological saline (1% NaCl). External lesions on the fish were sprayed with 70% ethanol, incised with an alcohol-flamed scalpel and sampled with a sterile inoculating loop. Fish body cavities were also observed for any abnormalities then the kidney and spleen were 20  aseptically sampled by cutting the organs with a sterile scalpel and swabbing them with sterile cotton-tipped applicators. Bacteriological sampling of sea lice followed Barker et al. (2009), including repeated sampling of disinfected lice prior to dissection to verify external disinfection; however, 90% ethanol (rather than 70%) was used for disinfecting specimens. All bacteriology samples were inoculated on Coomassie Brilliant Blue agar (CBB) (Udey 1982, Whitman 2004) at 22?C for 48-72 h. Colonies of typical A. salmonicida appear dark blue due to the blue protein dye in CBB absorbed by the bacterial A-layer, while the cells produce a brown, water-soluble pigment on the tryptone-containing agar (Austin and Austin 2007). Suspect colonies of A. salmonicida identified on CBB agar were restreaked for isolation on new CBB agar for 48 h at 22?C, Gram-stained, and identified using a variety of biochemical assays: oxidase, motility media supplemented with triphenyl tetrazolium salt for 48 h at 22?C, oxidative-fermentative (O-F) media for 48 h at 22?C, and API-20NE (BioMerieux?, verified by apiweb?) for 48 h at 22?C. 2.2.5 Sea Lice and Aeromonas salmonicida Challenges Hypothesis 1: Lepeophtheirus salmonis Can Acquire Aeromonas salmonicida from Water Bath Exposure. Adult, female sea lice (n = 10 per replicate) were placed in sterile aerated beakers containing 198 ml of ASW at 10?C and 2 ml of the appropriate A. salmonicida solution to produce various ten-fold concentrations (101- 107 cells ml-1) reflecting low ambient levels during epizootics (Rose et al. 1989). Lice were immersed in each concentration for three time periods (1.0, 3.0 or 6.0 h). Following removal from the baths, using sterile forceps, each individual louse was sampled (externally and internally) for the presence of A. salmonicida as noted above.   The highest bath concentration (107 cells ml-1) and exposure time (6 h) was then used to test longevity of the sea lice associated with A. salmonicida. After immersion at that 21  concentration and time, lice (n = 30 per beaker) were transferred to aerated, autoclaved beakers (3 replicates per sample time) containing ASW at 10?C in a walk-in refrigerator at VIU. At each 24-h time period (0-120 h post immersion), 5 lice from each replicate beaker were sampled externally and internally using methods as noted above. Every 8 h during the trial, lice were transferred into new autoclaved beakers containing ASW. This transfer was based on results of preliminary trials and was done to simulate a dilution effect that would occur in nature plus minimize A. salmonicida remaining as a biofilm on each beaker. Water quality measurements (as stated above) were taken at each sampling point. In addition, using sterile 5 mL pipettes, water samples (0.1 mL) from each beaker containing lice were plated on CBB and incubated at 22 ?C to quantify any bacteria residing in the water column.  Hypothesis 2: Lepeophtheirus salmonis Can Acquire Aeromonas salmonicida from Donor Fish via Parasitism. Generating A. salmonicida-infected sea lice through parasitism first required the production of A. salmonicida-infected hosts. In Trial 1 (24/3/2011-12/4/2011) Atlantic salmon (n = 30, mean wt = 44.5 g) were anaesthetized, ip-injected with 100 ?l of 4.5 ? 107 cfu mL-1 of A. salmonicida and exposed to sea lice (n = 188, ~6.27 lice fish-1) (Table 2.1). Atlantic salmon (n = 30, mean wt = 155.2 g) in Trial 2 (6/9/2011-27/9/2011) were also anaesthetized, ip-injected with 100 ?l of 5.54 x 107 cfu mL-1 of A. salmonicida and exposed to sea lice (n = 300, ~10 lice fish-1) (Table 2.1). These fish were referred to as ?donor? fish. Once sea lice had attached (~10-15 min), donor fish were placed into 236 L tanks (stocking density = 5.7 and 9.9 g L-1, flow rate = 8.4 L min-1) and checked daily for mortalities (Table 2.1). After 50% fish mortalities, remaining fish were euthanized (TMS 300 mg L-1) and sampled. Attached sea lice were removed, using sterile forceps, from dead and living donor fish and placed into a 1000 mL beaker with ASW for each trial. Sub-samples of the donor fish (Trial 1: n = 15, 50%; Trial 2: n = 16, 53.3%) and the 22  recovered sea lice populations (Trial 1: n = 5, 20%; Trial 2: n = 7, 7.2%) were tested via techniques described above for the presence of A. salmonicida. Sea lice not sampled were used in hypothesis 3. Hypothesis 3: Aeromonas salmonicida?exposed Lepeophtheirus salmonis Can Transmit the Pathogen to Recipient Atlantic Salmon Via Parasitism. Atlantic salmon used in Trials 1 and 2 (n = 24 each, mean wt = 46.0 g, 153.4 g, respectively) representing the recipient fish were anaesthetized, handled and exposed to sea lice (n = 20, 76) recovered from donor fish used in hypothesis 2 (Table 2.1). After sea lice attachment (10-15 min), the newly-parasitized recipient fish were relocated and equally divided (6 fish tank-1) into four replicate tanks in each trial. For Trials 1 and 2, we used four replicate 31 L tanks (8.6 g L-1, flow rate = 4.8 L min-1) and four replicate 62 L tanks (15.1 g L-1), respectively (Table 2.1). As a reference challenge control, an additional 24 Atlantic salmon in each trial (mean wt = 42.9 g, 157.0 g) were anaesthetized and handled as described above, then were exposed to 76 sea lice (~3.17 lice fish-1) (Table 2.1). After complete sea lice attachment (10-15 min), the parasitized fish were relocated (6 fish tank-1) into four replicate tanks per trial (Trial 1, 31 L, 8.6 g L-1; Trial 2, 62 L, 15.1 g L-1) (Table 2.1). Fish were checked daily for mortalities and monitored for 14 days post injection (dpi), after which, surviving fish were euthanized (300 mg L-1 TMS?). This time allowed for the development of A. salmonicida infections (if present). 2.2.6 Data Analyses Fish measurements were used to calculate Fulton?s condition factor [K = (weight / fork length3) ? 100], which describes the relative health ?condition? of each individual fish (Nash et al. 2006). We also recorded the numbers of sea lice feeding sites (FS) on host fish, characterized as small, round abrasions at typical sites of attachment (Pike and Wadsworth 1999). Correlation 23  coefficients (r) were examined between dpi versus cumulative fish mortality, and dpi versus K. Cumulative mortality (%) over experimental time was analyzed via regression models (R2).  The range of infection (R = the minimum to maximum number of lice per fish), prevalence (P = percentage of fish infected with lice or A. salmonicida), mean intensity (I = mean number of L .salmonis attached to infected salmon hosts in group) (Bush et al. 1997), and recovery percent (RP = number of recovered sea lice divided by the original number of sea lice used in infection) were calculated for each trial. Paired t-tests and independent samples t-tests were performed on fish condition factors before and after sea lice infections and between control and experimental salmon, respectively (NCSS 2007). The parametric one-way analysis of variance (ANOVA) was used to analyze differences among the mean number of sea lice feed sites (FS) and K per fish in challenge groups for both trials. The nonparametric Kruskal-Wallis one-way ANOVA was performed on data that did not meet the assumptions of normality, which included FS and K of recipient fish in Trial 2, and K of reference fish at the beginning of Trial 1. If an ANOVA was rejected, a post-hoc Tukey-Kramer multiple comparisons test was performed to identify which treatments differed. For all analyses, p < 0.05 was considered significant.        24  Table 2.1 Husbandry Conditions of Experimental Fish (Salmo salar) and Sea Lice (Lepeophtheirus salmonis) Density. Challenge Group n Fish  Weight (g) (?SD) Lice Density (lice fish-1) Stocking Density     (g L-1) Flow Rate (L min-1) Temp (?C) (?SD) Trial 1 (24/3/2011)             Donor 30 44.5 (10.3) 6.27 5.7 8.4 10.2 (0.5) Recipient 24 46.0 (12.5) 0.83 8.6 4.8 10.3 (0.6) Reference 24 42.9 (9.7) 3.17 8.6 4.8 10.1 (0.2) Trial 2 (6/9/2011)             Donor 30 155.2 (18.7) 10 9.9 8.4 11.1 (0.5) Recipient 24 153.4 (16.3) 3.17 15.1 4.8 11.0 (0.4) Reference 24 157.0 (26.2) 3.17 15.1 4.8 11.6 (1.6)  2.3 Results 2.3.1 Hypothesis 1: Lepeophtheirus salmonis Can Acquire Aeromonas salmonicida from Water Bath Exposure. Aeromonas salmonicida-positive sea lice were obtained by water bath immersion but there was a clear relationship between percent of lice testing positive and bacterial concentration. At a concentration of 104 cells ml-1 or higher, the percentage of lice positive for A. salmonicida on their external carapace ranged from 38.5% (105 cells ml-1) to 100% (107 cells ml-1) after 1.0 h exposure. A similar association was also observed with exposure time in which lice exposed to A. salmonicida for 3.0 or 6.0 h were more likely to test positive for the bacteria externally and internally. At these longer times, A. salmonicida was recovered from 20% (101 cells ml-1) to 100% (104- 107 cells ml-1) of lice. Lice were only positive for A. salmonicida internally when exposed to a concentration of 104 cells ml-1 or higher with the highest recovery (100%) at the highest concentration (107 cells ml-1) and longest exposure time (6.0 h). In the longevity assessments, 100% of the lice were positive for the bacteria externally up to and including 120 h 25  post immersion bath. Conversely, positive recovery of A. salmonicida residing inside the lice was lower (0-25%) and values were inconsistent among replicates. Plated water samples revealed a rapid decline of A. salmonicida: 82.67 ? 19.73 cells mL-1 at 24 h, 36.67 ? 5.77 cells mL-1 at 48 h, 3.33 ? 5.77 cells mL-1 at 72h, 0 cells at 96 h and 120 h. Water quality measurements were consistent throughout the experiment for pH (~8), oxygen (9.97 - 11.57 mg L-1) and temperature (9.1 - 9.53 ?C).  2.3.2 Hypothesis 2: Lepeophtheirus salmonis Can Acquire Aeromonas salmonicida from Donor Fish Via Parasitism. Trial 1 (24/3/2011 ? 12/4/2011) Aeromonas salmonicida-positive sea lice were generated through parasitism on A. salmonicida-infected Atlantic salmon. Fish mortality among the group of 30 donor fish parasitized by sea lice began at 4 dpi, and 24 h later, the cumulative mortality was 80% (24 of 30 fish). A sub-sample of 15 fish (50%, 3 moribund and 12 dead) tested positive for A. salmonicida (100%) from swabs of mucus, kidney and spleen (Table 2.2). The bacterial isolate was a non-motile, oxidase positive, fermentative, Gram-negative rod that produced dark blue colonies with brown water-soluble pigment on CBB agar. API-20NE resultant code (# 5574704) confirmed the isolate to be 98.5% A. salmonicida throughout all trials. External characteristics of fish diagnosed with A. salmonicida infections consisted of skin discoloration (darkening), slight exophthalmia, petechiae and haemorrhaging at the fin bases. Internally, fish exhibited darkening of the kidney, splenomegaly and haemorrhages over the abdominal walls. This A. salmonicida donor group was terminated on 5 dpi.  A total of 25 sea lice (recovery percent (RP) = 13.3%) were recovered from the dead and euthanized fish sampled on 4 and 5 dpi with the prevalence of lice infection and mean lice 26  intensity being 53.3% and 1.6 lice fish-1, respectively (Table 2.2). A sub-sample of the 25 recovered sea lice (n = 5, 20%) confirmed 100% internal and external presence of A. salmonicida (Table 2.2).  Trial 2 (6/9/2011 ? 27/9/2011) Mortalities among the donor fish (n = 30) parasitized by sea lice began at 6 dpi. The donor group was terminated at 7 dpi with a cumulative mortality of 53.3% (16 of 30 fish). Of the 16 mortalities, 100% tested positive for A. salmonicida from the mucus, kidney and spleen samples (Table 2.2). A total of 97 sea lice (RP = 32.3%, P = 90%, I = 3.6 lice fish-1) were recovered from the surviving fish (n = 14) after 7 dpi (Table 2.2). Among these 97 sea lice, a sub-sample of 7 (7.2%) tested positive for A. salmonicida (100%) on their external carapace, but there was no recovery of A. salmonicida from their internal stomach contents (Table 2.2).  2.3.3 Hypothesis 3: Aeromonas salmonicida?exposed Lepeophtheirus salmonis Can Transmit the Pathogen to Recipient Atlantic Salmon Via Parasitism. Trial 1 (24/3/2011 ? 12/4/2011) A total of 20 sea lice were recovered from the 30 donor fish (hypothesis 2, Trial 1) and were transferred among four replicate tanks holding 6 recipient fish each (~0.83 lice fish-1). Two fish escaped from one of the recipient tanks and were found dead with no A. salmonicida infections or sea lice present on day 6. These two fish were only included in the K data. The first fish mortalities of the remaining 22 recipient fish occurred at 8 dpi and continued to the end of the trial at 14 dpi (Fig. 2.1). The polynomial regression showed a positive relationship between cumulative percent mortality and dpi (y = 0.5338x2 ? 2.6205x + 0.8397, R2 = 0.9381, p < 0.001) (Fig. 2.1). However, there was no linear correlation between dpi and K of mortalities (r = 0.433, df = 12, p = 0.122) or dpi and fish mortality (r = 0.039, df = 11, p = 0.901). 27  The mean cumulative mortality of fish among the four replicate groups was 59.1% (13 of 24 fish) at 14 dpi (Table 2.2). Aeromonas salmonicida infections were detected from the mucus, spleen and kidneys of all 13 mortalities. On 14 dpi, the trial was terminated and the surviving fish (n = 9, 40.9%) were euthanized and sampled. From these survivors, 100% were positive for A. salmonicida in their mucus, 44.4% were positive in their kidneys, and 33.3% were positive in their spleen. When the data for mortalities and survivors were pooled, 100% (n = 22) were A. salmonicida positive from mucus, 77.3% (n = 17) from kidneys and 72.7% (n = 16) from spleen (Table 2.2). Three sea lice (two adult males at 10 dpi, one adult female at 14 dpi) were recovered (RP = 15%) from two dead and one moribund fish (Table 2.2). Among these recovered lice (P = 13.6%, I = 1.0 lice fish-1, FS = 1.1 ? 0.54 sites fish-1), 100% and 33.3% (one adult male) were positive for A. salmonicida externally and internally, respectively (Table 2.2).  There were no mortalities, A. salmonicida infections or recovered sea lice among the four replicate groups of reference fish infected with unexposed sea lice (FS = 2.2 ? 0.92 sites fish-1) during the 14 day experimental challenge (Table 2.2). There was a difference (H = 9.71, df = 3, p = 0.0212) in K among reference fish groups (Group 2 ? Group 3) at the beginning of the trial, but not after 14 d. Moreover, there was a difference (F = 4.38, df = 3, 20, p = 0.016) in FS among replicate groups (Group 2 ? Group 4). Temporally, there was no difference (t = 0.322, df = 6, p = 0.759) between mean K of the reference fish before and after sea lice infections (Fig. 2.2a). However, the mean K of the recipient fish group decreased (t = 5.29, df = 6, p = 0.00186) after sea lice infections (Fig. 2.2a). When comparing both treatments, there was no difference in mean K at the start (t = -0.245, df = 6, p = 0.815) and end (t = 2.37, df = 6, p = 0.0559) of the trial (Fig. 2.2a). Similarly, there was no difference in K and FS among the replicate groups of recipient fish.   28  Trial 2 (6/9/2011 ? 27/9/2011) Four replicates of 6 recipient fish each (n = 24) were infected with sea lice (n = 76, ~3.17 lice fish-1) recovered from donor fish (Hypothesis 2, Trial 2). No mortalities were observed among the four replicate recipient groups during the 14 day experimental challenge (Table 2.2). At 14 dpi, the trial was terminated and fish were sampled. All fish samples were negative for A. salmonicida (Table 2.2). A total of 17 L. salmonis (two adult males, 15 adult females) were recovered (RP = 22.4%) and sampled (Table 2.2). These 17 lice (P = 45.8%, I = 1.5 lice fish-1, FS = 1.3 ? 0.81 sites fish-1) tested negative for A. salmonicida externally and internally (Table 2.2).  There were no mortalities among the reference fish infected with unexposed sea lice and all tested negative for A. salmonicida (Table 2.2). At 14 dpi, nine sea lice (adult females) were recovered (RP = 11.8%) (Table 2.2). All nine lice (P = 33.3%, I = 1.1 lice fish-1, FS = 1.3 ? 0.75 sites fish-1) also tested negative for A. salmonicida externally and internally (Table 2.2). The four replicate groups of reference fish were equal in K and FS. Temporally, there was no difference (reference group, t = -2.33, df = 6, p = 0.0588; transfer group, t = 1.23, df = 6, p = 0.263) between mean K of fish before and after sea lice infections (Fig. 2.2b). Similarly, when comparing within treatments, there was no difference in mean K at the start (t = -0.943, df = 6, p = 0.382) and end (t = 2.20, df = 6, p = 0.0698) of the trial (Fig. 2.2b). There was no difference in K and FS among replicate groups of recipient fish.29  Table 2.2. Lepeophtheirus salmonis Acquisition of A. salmonicida After Parasitizing Bacteria Infected Atlantic Salmon (hypothesis 2) and A. salmonicida Transmission to Na?ve Atlantic Salmon Via Parasitism by A. salmonicida-exposed Sea Lice (hypothesis 3). Range of infection (R), prevalence (P), mean intensity (I), recovery percent (RP), and mean number of feeding sites (FS) of sea lice on Atlantic salmon. Percent of A. salmonicida-positive (A.sal+) sea lice with external (ext.) and internal (int.) infections and in Atlantic salmon mucus, kidney and spleen samples. dpi: days post-infection; CM: cumulative mortality.  Challenge treatment groups Trial No. of        fish dpi CM (%) Lepeophtheirus salmonis                        Mucus Kidney Spleen R P (%) I RP (%) FS     (? SD) A. sal+ ext. (%) A. sal+ int. (%) A. sal+ (%) A. sal+ (%) A. sal+ (%) Fish ip-injected with A. salmonicida and parasitized with unexposed sea lice 1 30 5 80 0-3 53.3 1.6 13.3 - 100? 100? 100 100 100 2 30 7 53.3 0-6 90.0 3.6 32.3 - 100? 0 100 100 100 Fish parasitized with A. salmonicida-exposed sea lice 1 22* 14 59.1 0-1 13.6 1.0 15.0 1.1 ? 0.54 100 33.3 100 77.3 72.7 2 24 14 0 0-4 45.8 1.5 22.4 1.3 ? 0.81 0 0 0 0 0 Fish parasitized with unexposed sea lice 1 24 14 0 0 0 0 0 2.2 ? 0.92 0 0 0 0 0 2 24 14 0 0-2 33.3 1.1 11.8 1.3 ? 0.75 0 0 0 0 0 *Two fish escapees at beginning of trial not used; ?Sub-sample of sea lice (Trial 1, n = 5; Trial 2, n = 7). 30   Figure 2.1. Hypothesis 3, Trial 1. Aeromonas salmonicida Transmission to Atlantic Salmon Via Bacteria-exposed Sea Lice. Mean (? SD) daily average cumulative percent mortality of S. salar among 4 replicate groups of recipient fish (fish parasitized with A. salmonicida-infected sea lice). All mortalities confirmed to have A. salmonicida infections in the mucus and kidneys (see text for details). Polynomial regression (y = 0.5338x2 ? 2.6205x + 0.8397, R2 = 0.9381, p < 0.001, n = 4).          0 20 40 60 80 100 0 2 4 6 8 10 12 14 Cumulative Mortality (%) Experimental Timeline (Days) 31      Figure 2.2. Mean (? SD) Fish Condition Factor Among Tanks (n = 4, 6 fish tank-1) of Reference Fish (Fish Parasitized with Unexposed Sea Lice) and Recipient Fish (Fish Parasitized with A. salmonicida-exposed Sea Lice) Groups Before (Day 0) and After (Day 14) Sea Lice Infections. a.) Trial 1. b.) Trial 2. Temporal differences between treatments were assessed using independent samples t-tests (p < 0.05). Significant temporal differences within groups are joined by a bracket and indicated by the Greek letter ?.    0.80 0.85 0.90 0.95 1.00 1.05 1.10 1.15 Condition Factor (K) 0.80 0.85 0.90 0.95 1.00 1.05 1.10 1.15 Day 0                            (n = 4)                                LEPs Day 14                           (n = 4)                       LEPs Day 0                              (n = 4)                                       A.sal & LEPs Day 14            (n = 4)       A. sal & LEPs Condition Factor (K) Reference Fish Recipient Fish ? a. b. 32  2.4 Discussion This is the first study to experimentally demonstrate the acquisition of Aeromonas salmonicida by Lepeophtheirus salmonis via water bath and parasitism and the successful transfer of bacteria from donor Atlantic salmon to recipient Atlantic salmon via parasitism. The acquisition of A. salmonicida by sea lice in a water bath is strongly influenced by concentration and exposure time. After the highest concentration (107 cells ml-1) and exposure time (6 h), viable A. salmonicida could be recovered from the louse carapace up to 120 h. Similarly, Jakob et al. (2011) reported dose and exposure time as key determinants of longevity of IHNV associated with sea lice; however, that association was more ephemeral (12-24 h). Interestingly, in the present study, bacterial water count measurements were reduced to zero while A. salmonicida was still recovered from 100% of the lice sampled externally (96 and 120 h). Aeromonas salmonicida has two strains: typical and atypical. The former represents the classic description of the bacteria and it contains an additional layer (A-layer) on the lipopolysaccharide (LPS) layer that has hydrophobic properties due to a high proportion of uncharged amino acids (Trust et al. 1983) and is correlated with cell virulence (Udey and Fryer 1978, Madetoja et al. 2003). This feature would favour A. salmonicida remaining associated with an organic, nonpolar surface (i.e., lice carapace). In the present study, A. salmonicida was still detected on lice after 5 d. Therefore, we hypothesize that sea lice can act as a substrate for this bacterium and increase its longevity in sterilized seawater.  The acquisition of bacteria by sea lice through parasitism appears to be related to fish size and time after death. Sea lice were collected from donor fish on the second day of mortalities in both trials, with mortalities occurring earlier in the Trial 1 smaller fish (mean wt = 44.5 ? 11.2 33  g). Sea ice recovered from the smaller donor fish contained 100% A. salmonicida on their external carapace and inside their digestive tract, while lice from the larger donor fish (mean wt = 155.2 ? 21.7 g) were 100% positive externally only. These differences may be due to larger fish exhibiting a slower development of disease because of a more developed immune system. The mortality of salmonids infected with IHNV was negatively correlated with fish age and size (LaPatra 1998, Bergmann et al. 2003). Theoretically, sea lice feeding on younger, more susceptible fish will have a higher probability of contacting and ingesting the pathogen because it would be present in higher numbers than lice feeding on larger, less susceptible fish. Interestingly, we were able to recover more sea lice off the larger fish (RP = 32.3%) than the smaller fish (13.3%). This could be a result of larger fish having a greater surface area to harbour higher lice numbers and larger fish would provide more nutrients and greater potential for survival. Lice that were recovered from parasitizing A. salmonicida-infected salmon were more likely to harbour A. salmonicida internally than those immersed in a concentrated bath. It appears that for L. salmonis to consistently acquire A. salmonicida internally, the lice may need to be actively feeding and therefore ingesting potentially contaminated materials (e.g., mucus).   Acquisition of bacteria by sea lice has been observed in previous studies. Barker et al. (2009) isolated three potentially pathogenic bacteria (Tenacibaculum maritimum, Pseudomonas fluorescens, and Vibrio spp.) from the external carapace and internal stomach contents of sea lice. Similarly, Nese and Enger (1993) isolated A. salmonicida from L. salmonis attached to Atlantic salmon suffering from furunculosis, suggesting the bacterium was residing inside the lice. Also, Jakob et al. (2011) sampled lice from Atlantic salmon (mean wt = 306.1 g) infected with IHNV on the second day of fish mortalities and observed 94% IHNV-positive sea lice, but did not differentiate the location (external or internal) of the virus associated with the lice. Their 34  observation of limited association time (24 h) suggests the virus does not replicate within lice, implying that lice are mechanical vectors. It is unclear if this hypothesis applies to bacteria. Nylund et al. (1991) suggested that pathogenic fish bacteria could survive and potentially multiply in the midgut of L. salmonis, especially when it is filled with partly digested epithelial tissue and blood from the host. This present study did not explore the potential of bacteria multiplication inside the sea louse L. salmonis. The question still remains whether L. salmonis act as a mechanical vector (source of accidental transport) or a biological vector (source of pathogen development and transmission)? Bacteria-positive lice did transmit A. salmonicida to recipient Atlantic salmon resulting in acute furunculosis and mortality. However, successful transmission did not occur in both challenges, which was likely dependent upon the complex interactions of host, pathogen and environment. For example, in Trial 1, A. salmonicida was transmitted to 100% and 77.3% (mucus and kidney infections, respectively) of recipient fish; whereas, in Trial 2 none of the recipient fish were A. salmonicida-positive. The transmission success observed in sea lice exposed to recipient fish in Trial 1 may be explained by the internal presence (100%) of A. salmonicida in L. salmonis prior to infection; but, lice in Trial 2 did not have A. salmonicida present internally. If true, then L. salmonis needs to feed on a host to act as a vector and transmit a pathogen. Similarly, Jakob et al. (2011) observed successful transmission of IHNV via L. salmonis when lice were able to attach to a host, but when lice were confined within a fine mesh box preventing attachment, virus transmission was not detected. Moreover, differences in host susceptibilities to A. salmonicida infections may exist between trials. Smaller and potentially more susceptible fish were utilized in Trial 1 compared to Trial 2. Atlantic salmon are among the most susceptible species to A. salmonicida and our observation of no infected fish in Trial 2 35  suggests either the bacteria were present in a very low concentration on the lice, the bacteria were unable to multiply inside/on the lice host and therefore died before transfer to recipient fish, lice need to internally obtain the pathogen to act as a vector for transmission or the larger fish used in Trial 2 were less susceptible to A. salmonicida. Water temperature and fish density are the only factors observed to have a significant impact in fish mortalities via cohabitation challenges with A. salmonicida (Nordmo and Ramstad 1999, Ogut and Reno 2004) and the water parameters for our challenges were similar.  The exact route of A. salmonicida transmission is ambiguous (Austin and Austin 2007). Probable routes of infection include: contact with infected fish, contaminated water or fish farm materials, transovarian transmission (McCarthy 1980), ingestion of contaminated food (Burger and Bennett 1985), horizontal or vertical transmission via carrier fish, which show no signs of disease but harbor the pathogen (Austin and Austin 2007) and vector transmission via sea lamprey (El Morabit et al. 2004). Similarly, the precise site(s) of entry into a host is still uncertain with possible portals of entry through the gills, lateral line, mouth, anus or injury (Effendi and Austin 1995). Clearly, A. salmonicida is not dependent on sea lice for transmission like Plasmodium requires a mosquito vector. Variable time periods have been observed regarding A. salmonicida survival in the environment, with survival in unsterilized seawater between 24 h and 8 days (McCarthy 1980). Moreover, fish exhibit a stress response (i.e., increased cortisol levels and decreased macrophage activity) when infected with sea lice (Nolan et al. 1999, Pike and Wadsworth 1999, Bowers et al. 2000, Fast et al. 2006), which chronically may lead to host immunosuppression and higher susceptibility to various pathogens (Pickering and Pottinger 1989, Bowers et al. 2000). Cusack and Cone (1986) hypothesized that a parasite vector may offset a low transmission efficacy of a pathogen, particularly a pathogen with short 36  survival times outside the host. Thus, a high infectious dose carried by the ectoparasite L. salmonis could play a role in bacteria dissemination and longevity in the marine environment. More studies are required to test the hypotheses that sea lice infections would increase transmission success of A. salmonicida. Fish condition factor (K) was affected by A. salmonicida infections in the presence of sea lice. In Trial 1, recipient fish that were exposed to lice and bacteria showed a decrease (t = 5.29, df = 6, p = 0.00186) in K after the introduction of A. salmonicida-carrying sea lice. Conversely, there was no difference (t = 1.23, df = 6, p = 0.263) in the K of recipient fish in Trial 2. There was also no difference (t = -0.741, df = 6, p = 0.485) in mean lice feed sites (FS) between transfer fish in each trial. These data suggests the difference in mean fish K among trials is associated with synergistic effects of L. salmonis and A. salmonicida infections rather than being solely impacted by the amount of sea lice feeding. Similarly, Jones and Nemec (2004) concluded that natural infections of L. salmonis did not affect the size or the K of pink and chum salmon (up to ~6 g). However, when chum salmon were exposed to high lice infections (735 copepodids per fish), fish weight was significantly less than that of unexposed fish (Jones et al. 2007). The presence of an ectoparasite and a bacterial pathogen may trigger a stress response and consequently a low appetite in host fish, resulting in loss of fish weight and K. Most of the fish exhibited symptoms (discoloration, scale loss and lesions) typical of L. salmonis infections (J?nsd?ttir et al. 1992, Grimnes and Jakobsen 1996, Johnson et al. 1996). Sea lice feeding sites (FS), which typically leave an ?oval? imprint corresponding to the margin of the parasite cephalothorax (J?nsd?ttir et al. 1992), were present on most fish. Enumerating FS may have errors since lice sometimes wedge themselves under the scales of fish leading to a damaged epithelium that becomes covered by the scales when the parasite leaves the site. 37  Moreover, sea lice need to feed on an area for an unspecified time to elicit an observable FS. Despite such potential errors, we wanted to include analysis of FS that were prominent and indicative of parasite feeding because blood consumption by lice would have occurred. Thus, the presence of these FS supports the hypothesis that sea lice feeding behavior on a host provides portals of entry for secondary infections.  Results from this study indicate that preadult and adult Lepeophtheirus salmonis are able to acquire and transmit Aeromonas salmonicida to recipient fish through parasitism under specific experimental conditions. However, it appears L. salmonis need to orally obtain the pathogen to successfully transmit A. salmonicida to small fish presumably via parasitism. A large inoculum of A. salmonicida (107 CFU mL-1) was used in these challenges. The probability of A. salmonicida occurring in these numbers in the natural environment is unlikely, except perhaps during a severe epizootic event where moribund and dead fish were releasing large numbers into the immediate vicinity and with low dilution effect of the water (Austin and Austin 2007). Given the highly hydrophobic and obligate pathogenic characterisitics of A. salmonicida, there is potential for L. salmonis to act as a biological vector and transmit the pathogen to young, na?ve Atlantic salmon.       38  3 General Discussion 3.1 Sea Lice Acquisition of A. salmonicida The potential of adult Lepeophtheirus salmonis to acquire Aeromonas salmonicida from ip-injected (?donor?) Atlantic salmon and transmit the pathogen to uninfected (?recipient?) Atlantic salmon via parasitism was examined under laboratory conditions. In the present study, the mortality rates of the donor fish (Fig. A.1) were similar to Nordmo et al. (1998), who observed ip-injected (5 x 104 CFU of A. salmonicida) S. salar (25-75 g) mortalities beginning 4 to 6 dpi and reaching 90% cumulative mortality within 11 dpi. The high cumulative mortalities of ?donor? fish observed in the current study provides further evidence of the high pathogenicity of A. salmonicida, particularly for young S. salar at 44 and 155 grams. Concentrated (105-107 cfu mL-1) ip-injections of A. salmonicida have similar effects on S. salar of different sized groups up to 155 g (pers. obs.). However, the virulence of A. salmonicida in host fish and the ability of sea lice to obtain the pathogen from such a host via parasitism appear to depend on fish size and time after death. Aeromonas salmonicida was present externally on lice and it can be assumed the pathogen was present in the fish mucus and water column via fish shedding the bacterium. However, the amount of bacteria being shed into the water column was not enumerated. Moreover, the exact route of A. salmonicida shedding by host fish is inconclusive. Aeromonas salmonicida has been hypothesized to be transmitted from host fish via furuncles (up to 108 viable cells mL-1), muscle tissue (McCarthy, 1977), mouth, vent and injection point. Due to the early mortalities (acute infection) of host fish in the present study and lack of furuncles, the vent, mouth, mucus and injection point are the most likely sources of infective bacteria.  39  Aeromonas salmonicida is an obligate pathogen that has a hydrophobic surface. It is highly probable that the bacteria, once extruded by the host, will attach to any organic object in the water column such as the mucus of fish and the external carapace of L. salmonis. The presence of A. salmonicida in the surrounding water column does not necessarily mean the lice will obtain the pathogen internally. The lice need to consume the bacteria via accidental ingestion and the bacteria need to survive and persist while associated with the parasite. The presence of A. salmonicida internally in sea lice from donor fish suggests the pathogen was obtained via feeding on the host (Fig. A.2). Conversely, the absence of A. salmonicida internally in sea lice from Trial 2 donor fish could be explained by the inability of the bacterial cells to grow on the CBB agar (e.g., cell numbers were well below detectable limits), the immunocompetence of the host fish or sampling limitation (e.g., swab did not obtain bacteria). Given the fact that A. salmonicida was detected in the external samples of lice and the pathogen was a reliable strain, suggests the medium was adequate and the cells will grow on CBB. Thus, competitions with non-pathogenic bacteria, sampling limitations and/or minute concentrations of A. salmonicida in the lice samples are likely possibilities. Another potential explanation could be that larger fish tend to exhibit a slower development of disease compared to smaller fish because of a more competent immune system. There is a negative correlation between mortality of salmonids infected with infectious haematopoietic necrosis virus (IHNV) and fish size and age (LaPatra, 1998; Bergman et al., 2003). Namely, in the present study, the smaller donor fish had a higher cumulative mortality compared to the larger fish. It seems that younger, more susceptible fish will succumb to disease and shed the pathogen earlier (and likely in greater concentrations) than larger fish, which in turn can lead to an increased probability of acquisition of the pathogen by feeding parasites (i.e., L. salmonis). 40  Cusack and Cone (1986) hypothesized that a low transmission efficiency of a pathogen, particularly an obligate pathogen with short survival times outside the host (i.e., infectious pancreatic necrosis virus and A. salmonicida), may be offset by a parasite vector. The dissemination and longevity of a bacterial pathogen in the marine environment may be increased when carried by a parasite vector (i.e., L. salmonis). This could have potential implications at marine net pen fish farms because the introduction of parasites from wild fish could concomitantly involve the introduction of fish pathogens, or vice versa (Cusack and Cone, 1986). Previous studies have isolated bacteria from sea lice. Three potentially pathogenic bacteria (Tenacibaculum maritimum, Pseudomonas fluorescens, and Vibrio spp.) were isolated from the external carapace and internal stomach contents of preadult and adult sea lice collected from healthy farmed Atlantic salmon around Vancouver Island, BC, Canada (Barker et al., 2009). The prevalence of bacteria has been reported to be higher during the higher water temperatures in the summer months and among adult stages (Barker et al., 2009). In Norwegian salmon farms, A. salmonicida has also been isolated from surface-disinfected (70% ethanol) L. salmonis attached to Atlantic salmon with furunculosis, suggesting lice harboured the bacteria in their gastrointestinal tract (Nese and Enger, 1993). Jakob et al. (2011) observed 94% of the sea lice to be positive for infectious haematopoietic necrosis virus (IHNV) when sampled from IHNV-infected Atlantic salmon on the second day of fish mortalities. Unfortunately, the authors failed to determine whether the virus was located externally or internally among the lice. However, they did observe virus association with lice was limited to 24 h and viral titres rapidly diminished, suggesting IHNV does not replicate while associated with sea lice. Petterson et al. (2009) could not determine if salmonid alphavirus SAV3, causative agent of pancreas disease in Atlantic salmon, could replicate within lice. Consequently, Jakob et al. (2011) hypothesized L. 41  salmonis acts as a mechanical vector for IHNV. However, this hypothesis may not be applicable for some bacteria, particularly A. salmonicida. Nylund et al. (1991) found bacterial rods in the midgut lumen and in different types of midgut cells of L. salmonis. They suggested that pathogenic fish bacteria have the potential to survive and multiply inside the midgut of L. salmonis, especially when it is filled with partly digested epithelial tissue and host blood. The present study did not investigate the potential bacterial multiplication of A. salmonicida within L. salmonis, but the bacterial pathogen was identified in internal samples taken after external disinfection (90% ethanol) from sea lice collected off infected fish. Further research needs to explore whether L. salmonis acts as a mechanical (source of accidental transport) or a biological vector (source of pathogen development and transmission) of A. salmonicida.  3.2 Aeromonas salmonicida Transmission Via Sea Lice Aeromonas salmonicida was transmitted to recipient fish via bacteria-exposed L. salmonis in the first trial of this study. Recipient fish that became infected exhibited small haemorrhages at their fin bases and over their abdominal walls, skin darkening (melanosis), enlarged spleen (splenomegaly) and darkening of their kidneys resulting in rapid mortality, typical of acute furunculosis. However, the precise mechanism of pathogen transfer was not explored. Successful transmission may be dependent upon the complex interactions of host, pathogen, and environment. Bacterial transmission success was most likely associated with the presence of the pathogen internally in the sea lice, the intensity of the lice feeding site and the fish size/age. In the present study, sea lice that were harboring A. salmonicida internally were able to transfer the pathogen to recipient fish, while lice not harboring internal bacteria were unable to transfer the bacteria. The mouth structure and mode of feeding exhibited by the ectoparasitic grazing of L. salmonis has the potential to facilitate oral transmission of bacteria 42  (Barker et al., 2009). Mucus and secretions from Atlantic salmon and rainbow trout (O. mykiss) resulted in a high proportion of lice releasing multiple types of low molecular weight proteases (Fast et al., 2003). Adult L. salmonis have a short tubular foregut and a large wide midgut that are lined by cuticle and separated by a pair of valves (Bron et al., 1993). Bacteria lining such a simple gastrointestinal tract could easily enter and exit the louse, especially during feeding and the discharge of enzymes. Jakob et al. (2011) failed to show that the presence of IHNV-infected L. salmonis in a fish tank was enough to cause infections among na?ve fish in the same tank. Successful transmission of IHNV from louse to fish only occurred when lice were allowed to attach to a host fish, not when lice were confined and prevented from attaching.  During Trial 2 of the present study, a similar pilot experiment was performed to test the potential of passive transmission via lice to fish. Aeromonas salmonicida-exposed sea lice (n = 14) from Trial 2 donor fish were individually rinsed with sterile saline and placed in an isolated net-cage inside a 62 L tank containing recipient fish (n = 3) for 14 days. Following this incubation period, we failed to provide evidence of the pathogen in/on fish. Although this pilot experiment was too small to be statistically relevant, it reflects the observations of Jakob et al. (2011). Lack of fish mortality and the highly hydrophobic properties of A. salmonicida may best explain the lack of passive transmission. Aeromonas salmonicida cells will naturally stick to nonpolar objects than remain in the water column. In both exclusion experiments above, physical contact between lice and fish was prevented. Presumably, bacterial cells remained associated with the lice.  Wikel et al. (1996) hypothesized that arthropod-mediated host immunosuppression can provide a favourable environment for the establishment of vector-borne pathogens. The feeding behaviour of L. salmonis can lead to a stress response in the host fish (i.e., increased cortisol, 43  decreased macrophage function) (Nolan et al, 1999; Pike and Wadsworth, 1999; Bowers et al., 2000; Fast et al., 2006). The resulting immunosuppression of the host increases susceptibility to other pathogens (Pickering and Pottinger, 1989; Bowers et al., 2000) and the lice feeding sites provide potential portals of entry for those pathogens. Sea lice are described primarily as skin grazers that ingest food opportunistically through epidermal damage (Pike and Wadsworth, 1999), opposed to true blood feeders such as leeches. Aquatic leeches perform multiple host switches and are known vectors for the pathogens Trypanoplasma borelli and Cryptobia (Trypanoplasma) salmositica (Becker and Katz, 1965; Kruse et al., 1989). Adult male and female L. salmonis do occasionally feed on blood, which was observed in my pre-trials (Fig. A.2) and has been identified spectrophotometrically in gut contents (Brandal et al., 1976). Lice that are able to feed on host blood would have a greater potential to act as vectors.  Host susceptibilities to A. salmonicida infections may have been different between the two trials. In Trial 1, smaller (and potentially more susceptible) fish were used compared to fish in Trial 2. It is hypothesized that younger fish have a less developed immune system than their older counterparts. Larger fish have a greater volume relative to surface area that reduces the impact of multiple lice feed sites on the fish. Whereas, the same number of lice feed sites on a smaller fish could have detrimental effects. For A. salmonicida, the size and age of fish have not been implicated as important factors affecting its virulence because Atlantic salmon are a very susceptible species. Studies have indicated that water temperature and fish density can impact salmon mortalities in cohabitation challenges with A. salmonicida (Nordmo and Ramstad, 1999; Ogut and Reno, 2004). Ogut and Reno (2004) tested chinook salmon (~1.7 g) and observed the prevalence and disease-specific mortalities from furunculosis to be considerably higher (>90%) at the highest stocking density (15.52 g fish L-1) than at the lower densities. Conversely, fish 44  density (15.1 g L-1) in Trial 2 of the present study was higher than that in Trial 1 (8.6 g L-1), yet no fish developed disease in Trial 2. Thus, the potential synergistic impact of sea lice and A. salmonicida infections affecting transmission efficacy in fish needs to be further studied. Aeromonas salmonicida is an obligate pathogen that has properties which enable it to have many transmission routes (Austin and Austin, 2007). One potential route is via the ingestion of contaminated food. This process begins with the movement of A. salmonicida cells from water into the surface microlayer via rising gas bubbles (Burger and Bennett, 1985) and by lipid droplets from lipid rich food or faecal material (Austin and Austin, 2007). Food pellets then penetrate the air-water interface, get covered with the surface microlayer and bacteria within, and eventually get eaten (Burger and Bennett, 1985). The surface microlayer is an important area for the aggregation of hydrophobic bacteria. Enger et al. (1992) found the total A. salmonicida counts to be one order of magnitude higher in the surface microlayer than corresponding counts in samples 10 cm below the water surface. McCarthy (1980) reported contact with infected fish, contaminated water or fish farm materials and transovarian transmission to be all probable routes of infection. Enger et al. (1992) observed a more than 10 fold decrease in the number of A. salmonicida cells in the surface microlayer when dead infected fish were removed from tanks. Another potential route of infection is horizontal or vertical transmission via carrier (or latent carrier) fish, which are hosts that show no disease but harbour the pathogen (Austin and Austin, 2007). Carrier fish are presumed to act as reservoir hosts, retaining the pathogen in fish populations (Austin and Austin, 2007). If cohabitation and bath challenges mimic natural exposure to A. salmonicida (Bricknell, 1995), then introducing a natural ectoparasite (i.e., L. salmonis) that has been exposed to A. salmonicida to na?ve Atlantic salmon should also serve as a suitable infection model.  45  The precise site(s) of pathogen entry into a host fish by A. salmonicida is unclear. Possible portals of entry may be through the hosts? gills, lateral line, mouth, anus or a surface injury (Effendi and Austin, 1995). Aeromonas salmonicida is not dependent on sea lice for transmission but its dispersion may be facilitated by lice.  Sea lice infections were produced by using the technique developed by Hull (1997) and Hull et al. (1998), which involves placing lice in a water column near a host fish and allowing it to attach without intervention. Hull et al. (1998) observed resettlement rates of over 80% when the parasite was separated from the host for less than 24 h, with the most consistent results occurring with separation times less than 30 min. In the present study, separation times were under 24 h and the recovery of L. salmonis among challenge treatment groups ranged from 0% to 32.3%. There are several possible reasons for these low values. First, lice were collected from dead, moribund and living fish multiple days after infection, which would have given the lice plenty of time to detach from hosts and search for a new one. Second, tanks provide a limited amount of space and the walls act as barriers. Fish can constantly bump and rub against the tanks which would result in removal of ectoparasites. Third, lice that detached from fish may have reattached to tank walls or been eaten by fish. Also, lice age was unknown and thus, natural mortality independent of any host response is possible. Lastly, lice may have rejected the host fish used in the trials. Sea lice were collected from larger/older Atlantic salmon and transferred to smaller/younger salmon. Lice were acclimated to the physiology and behaviour of larger, farmed fish. Moreover, lice were collected from fish in open-net pens in the ocean and then placed in to a laboratory environment (consult Table A.1 for water parameters). Also, lice were handled prior to the trials and this could have contributed to low lice survival. The copepods were collected using forceps from living adult salmon that had been sucked up through a large 46  pipe onto a harvesting rack, (where the host fish were handled and killed). Then, the lice were placed into aerated buckets, transported via boat and vehicle and placed on experimental fish within 24 h. Consequently, the infection success of L. salmonis from large Atlantic salmon to smaller Atlantic salmon should be explored in future research.  The number of sea lice feed sites (FS) on salmon from reference and recipient groups after infections in both trials were examined (Fig. A.3). The Trial 1 reference fish had greater FS (t = -3.10, df = 6, p = 0.0212) than that of Trial 1 recipient fish and Trial 2 reference fish (Fig. A.3). The FS data may not represent a precise measurement of lice feeding/attachment sites, but it is a more accurate assessment of lice activity than lice intensity data. Limitations of feed site data include areas not readily visible (e.g., sites under fish scales and sites of short term feeding). Despite the mean FS difference between groups in Trial 1, K appears to be more affected by the type of A. salmonicida infections. There was a decrease in the K of Trial 1 recipient fish after the introduction of A. salmonicida-exposed sea lice, but there was no difference in the mean K of reference fish (Fig. 2.1). Natural infections of L. salmonis do not affect the size or K of pink and chum salmon (up to ~6 g) (Jones and Nemec, 2004); whereas, large lice infections (735 copepodids fish-1) on chum salmon (20.0 ? 1.2 g) can decrease (p < 0.01) fish weight (Jones et al., 2007). Alternatively, ectoparasitic arthropods can down-regulate host innate and specific acquired immune defenses (Wikel et al., 1996; Braden et al., 2012). Moreover, ionregulation is an important homeostatic process of all teleost fish. Brauner et al. (2012) observed a significant ionic burden (increase in total body Na+) in tiny pink salmon (0.2 - 0.4g in weight) to be triggered by sea lice infections as the lice approached the preadult stage. The presence of a grazing ectoparasite and pathogenic bacterium may lead to osmoregulatory stresses on small fish and consequently lead to loss of fish weight or ?robustness? (= lower K value).  47  3.3 Pilot Experiment Histopathological examination of fish infected with lice demonstrated a localized inflammatory response beneath the site of lice attachment (Fig. B.1). Consequently, fish that were infected with A. salmonicida and lice exhibited a more widespread inflammatory response (Fig. B.2). Spongiosis, the separation of individual malpighian cells by tissue fluid, is an early indicator of an inflammatory response (Roberts, 1978). Ulcerations in the epidermis allow the infiltration of opportunistic pathogens, and cause osmotic stress (Roberts, 1978). Toxin production by Gram-negative bacteria such as Aeromonas spp., Pseudomonas spp. and Vibrio anguillarum can lead to karyolitic lesions (globules of deeply basophilic nucleic acid) (Andr? et al., 1972; Roberts and Bullock, 1976). The thickening of mucus layer in samples may be due to the absence of scales. Moreover, coccobacillus-shaped bacteria typical of young A. salmonicida cells were present in the epidermal and dermal layers (Fig. B.2). Similarly, Johnson and Albright (1992) examined the histological response of Atlantic, chinook and coho salmon to copepodids of L. salmonis and observed minor gill and fin tissue response among Atlantic salmon. The histological responses of my pilot study provide further evidence that L. salmonis has the potential to acquire bacteria via feeding on ip-injected fish. Jones et al. (2008) concluded that histological changes in pink salmon skin coincided with fish growth, which included thickening of the epidermis, infiltration of the dermis with fibroblasts and the first evidence of scales. It appears the mechanisms by which juvenile Oncorhynchus spp. avoid the effects of severe L. salmonis infection is associated with inflammation at the site of attachment, as well as the local and systemic elaboration of proinflammatory cytokines  (Johnson and Albright, 1992; Fast et al., 2002). In contrast to the present study, Jones et al. (2008) concluded susceptible Atlantic salmon to not elicit an inflammatory reaction at the site of lice infection. It is important to note that they 48  used the copepodid lice stage whereas I used the adult stage in my study. The present study revealed that adult sea lice in the presence of a pathogenic pathogen can lead to a widespread inflammatory response in young Atlantic salmon.  3.4 Serendipitous Discoveries Experimental research can be very tedious, stressful and exciting and can sometimes lead to unexpected surprises. Prior to my thesis, I did one year of experimental research with sea lice, A. salmonicida and salmon. During this time, I performed some pilot experiments examining sea lice acquisition of bacteria via bath exposure, cohabitation with infected fish and dead fish. Three trials were actually performed for this thesis, but the original Trial 2 was terminated from the manuscript due to a Pseudomonas fluorescens infection (one causative agent of fin rot) in the test population of fish. Consequently, the test population of fish needed to be treated, tanks needed to be sterilized and a new sea lice collection date needed to be arranged. This unexpected event is the reason for the large size/weight difference between Trial 1 and 2 fish described in Chapter 2. In addition, there were other serendipitous discoveries. One surprise was the identification of Kudoa thyrsites (Myxozoa) in the musculature of three reference fish from Trial 2 (Fig. C.1). This myxosporean parasite is known for forming macroscopic cysts in the host muscle and causing post mortem tissue degradation (Moran et al., 1999). The softening of fish flesh is most likely due to the release of proteolytic enzymes by the parasite (Tsuyuki et al., 1982). This condition presents a problem for salmon farmers since it makes the post-harvest fillet undesirable to the customer, leading to loss in revenue. Another exciting surprise was the observation of the red algae species Antithamnion growing on the carapace of L. salmonis (Fig. C.2). This form of hyperparasitism between algae and animal has never been documented for L. salmonis. This epiphytic algal species is highly opportunistic and will attach to stationary substrates (O?Clair et 49  al., 2001). The fact that it was growing on sea lice, suggests the lice came from an environment with very low water current. Both surprises were unexpected since little is known regarding K. thyrsites transmission and hyperparasitism with sea lice. 3.5 Future Research This study produced new questions and hypotheses that certainly warrant further investigation, for instance: 1. Does size of Atlantic salmon influence the amount of A. salmonicida shed, and consequently, lice acquisition of the pathogen? Examination will require different-sized groups of fish ip-injected with A. salmonicida and infected with sea lice. Samples (from different water column depths, tank surface, sea lice and fish) will need to be collected systematically (every hour, two hours, twice a day, etc.) over time leading to the last fish mortality.      2. Can Aeromonas salmonicida replicate within the sea louse L. salmonis? This study will need to examine lice with A. salmonicida internally and lice with bacteria and epidermal particles (from fish host) internally. It would be useful to externally disinfect lice samples and then run them through qPCR, supplemented with histology.   3. Do adult L. salmonis that have limited their parasitism to one species of salmon prefer a host of the same species and age? Experimental design requires the use of different salmon species and ages cross-infected with lice. In our preliminary trials (unpublished data), we did note reduced lice infections if the lice originated from a different source 50  than that used for the experiment (i.e., collected from wild Pacific salmon then transferred to cultured Atlantic or from cultured Atlantic to cultured Pacific).   4. Do adult female lice have a greater success rate of pathogen acquisition from host fish compared to adult males? This hypothesis is based on the evidence that adult male lice are more active on the surface of their host and are thus far more likely to transfer to new hosts as they actively seek females to mate (Ritchie, 1997). Males may therefore be more suitable candidates as vectors, but concurrently may not be feeding long enough on hosts to obtain pathogens.  5. Is there a positive correlation between the density of pathogen-carrying sea lice and pathogen transmission to host fish? An experimental study using different sea lice densities should be performed.  6. Is L. salmonis a suitable indicator species of environmental pathogens? A study examining spatial-temporal differences in pathogens collected by lice should be performed. Lice samples would need to be collected from different regions over different time periods. This research was begun by Barker et al. (2009), but the sample area needs to be greater and examined throughout the year.     7. What are the sequential histological effects of sea lice feeding and A. salmonicida infections on salmon skin. This study should examine variables such as time after infection, infectious dose, and salmon species. The knowledge gained from this work 51  would help better understand the relationship between louse and pathogen infections simultaneously.   8. Can sea lice act as vectors for other pathogens? Studies could examine the vector potential of sea lice with different bacterial species (e.g., motile species such as Vibrio or Tenacibaculum). 3.6 Research Implications The sea louse, Lepeophtheirus salmonis, can act as a vector of the bacterium, A. salmonicida by transferring it to young Atlantic salmon (~ 44g). However, it should be noted that the bacterial concentration used to inject donor fish was very high (107 CFU mL-1) and would not occur in nature unless there was a severe epizootic resulting in high morbidity and the release of large numbers of A. salmonicida into the immediate vicinity. In addition, parameters such as very low water exchange, the presence of unvaccinated fish and high temperatures (> 10?C) would be required to promote high bacterial concentrations. The concentration of bacteria was not measured on or in the sea lice, so it is unclear how much bacteria was exposed to recipient fish. If pathogens can persist inside lice and lice can transmit them, then there will always be the potential for transmission and dispersal regardless of pathogen concentration.  Lepeophtheirus salmonis has the potential to transmit pathogens to other fish species, other than salmonids. Aeromonas salmonicida has been isolated from wrasse (Labridae spp.), which are used as cleaner fish to eliminate sea lice in salmon farms (Treasurer and Cox, 1991; Laidler et al., 1999). Wrasse can obtain A. salmonicida infections via cohabitation with salmon in cages (Treasurer and Cox, 1991; Treasurer and Laidler, 1994), but I would hypothesize that 52  wrasse would also have the potential to obtain infection via feeding on infected sea lice. However, wrasse (especially Ctenolabrus rupestris) are more resistant to A. salmonicida infections than Atlantic salmon and are more likely to act as a reservoir (Bricknell et al., 1996).  My research may be useful for future fish health management implications of sea lice and the relationship between farmed Atlantic and wild Pacific salmon on the western coast of North America. Such implications should be considered for future planning, pathogen surveillance/monitoring and policy making and implementation. Currently, sea lice control is an integral part of management strategies for the control of the viral disease, infectious salmon anemia (ISA) in Atlantic Canada and Scotland (Johnson et al., 2004). Since 2003, a very rigorous and extensive sea lice management strategy has been underway in British Columbia. The overall goal is to help mitigate/prevent risks from serious pathogens. Hopefully, this research may motivate further investigations on the vector potential of sea lice in salmon aquaculture systems.  Little research has been conducted on the association between L. salmonis and accompanying pathogens. For several years, it was hypothesized that lice had the potential to act as vectors of fish pathogens. Jakob et al. (2011) were the first to experimentally test this hypothesis and concluded L. salmonis to be an ?accidental?, mechanical vector of IHNV. My study parallels Jakob et al. (2011) and it is the first to examine lice association with a bacterial pathogen. Both studies have revealed that sea lice associated with a fish pathogen have the ability to transmit that pathogen via parasitism under a laboratory setting. In the present study, it was further observed that the condition factor of fish parasitized with sea lice and infected with A. salmonicida decreased within the trial time. Unfortunately, the exact route of pathogen transmission and the vector category (biological or mechanical) remains unresolved. Lice can 53  obtain A. salmonicida from infected fish, but it appears internal acquisition of the bacteria is related to fish size, lice feed site intensity (blood feeding) and time post-mortem. I believe that a high concentration of the hydrophobic, pathogenic A. salmonicida in nature will attach to any nonpolar object (preferably organic) in the water and thereby increase its potential for transmission and dispersal, regardless if a copepod vector is present. 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Finfish and shellfish bacteriology manual: techniques and procedures. Iowa State Press, Iowa. Wikel, S. K., R. N. Ramachandra, and D. K. Bergman. 1996. Arthropod modulation of host immune responses. In The Immunology of Host-Ecto-parasitic Arthropod Relationships. Ed. Wikel, S. K., CAB International, Wallingford, UK. P. 107-130.  Yazawa, R., M. Yasuike, J. Leong, K. R. von Schalburg, G. A. Cooper, M. Beetz-Sargent, A. Robb, W. S. Davidson, S. R. Jones, and B. F. Koop. 2008. EST and mitochondrial DNA sequences support a distinct Pacific form of salmon louse, Lepeophtheirus salmonis. Marine Biotechnology 10: 741-749.            65  APPENDICES Appendix A: Supplemental Data  Figure A.1 Cumulative Daily Percent Mortalities of S. salar Ip-injected with A. salmonicida (Trial 1: 4.5 ? 107 cfu mL-1; Trial 2: 5.54 ? 107 cfu mL-1) and Infected with Unexposed L. salmonis (Trial 1, n = 188; Trial 2, n = 300). All fish mortalities confirmed to have acute furunculosis (see text for details).  Figure A.2 Lepeophtheirus salmonis Feeding on an Atlantic Salmon (S. salar). Note fish blood lining the digestive tract of louse. (Photo: C. Novak)  0 20 40 60 80 100 0 1 2 3 4 5 6 7 Cumulative Mortality (%) Experimental Timeline (Days) Trial 1                                                    (n = 30) Trial 2                                                     (n = 30) 66  Table A.1 Mean (? SD) Seawater Parameters in Tanks Containing Treatment Fish Groups in Trials 1 and 2. Water temperature (T), salinity parts per thousand (0/00), dissolved oxygen (D.O.), days post-infection (dpi) and standard deviation (SD). Fish treatment groups Trial dpi T (oC)       Salinity (0/00)  D. O. (mg/L)  pH  (? SD) (? SD) (? SD) (? SD) Donor fish infected with na?ve sea lice 1 5 10.3 (0.7) 30.2 (0.4) 10.8 (0.2) 7.8 (0) 2 7 11.1 (0.5) 30.4 (0.5) 10.1 (0.2) 8.1 (0.04) Recipient fish infected with A. salmonicida-positive sea lice 1 14 10.5 (0.6) 30.5 (0.7) 11.0 (0.2) 7.9 (0.07) 2 14 11.0 (0.4) 30.0 (0) 10.3 (0.07) 8.2 (0.05) Reference fish infected with na?ve sea lice 1 14 10.1 (0.2) 30.3 (0.6) 11.4 (0.2) 7.9 (0.05) 2 14 11.6 (1.6) 30.1 (0.3) 9.7 (0.3) 8.2 (0.08)    Figure A.3 Mean (? SD) Number of L. salmonis Feed Sites (FS) on Atlantic Salmon in the Reference (Fish Parasitized with Unexposed Lice) and Recipient (Fish Parasitized with A. salmonicida-positive Lice) Groups After Sea Lice Infections for Each Trial. Differences among challenge groups and trials were determined using a one-way ANOVA (p < 0.05). Tukey-Kramer comparison test was performed to determine which group differed (p < 0.05). Differences between challenge groups within each trial were determined using independent samples t-tests (p < 0.05). The asterisk (*) represents the treatment significantly different from the other three. Significant differences within trials are joined by a bracket and indicated by the Greek letter ?. The n-values represent the number of tanks containing 6 fish each.  0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 Lice                         (n = 4) Bacteria & Lice                 (n = 4) Lice                               (n = 4) Bacteria &Lice                          (n = 4) FS (sites fish-1) Trial 1                                        Trial 2 ? * 67  Appendix B: Pilot Study Atlantic salmon (n = 18, 46.0 ? 12.5 g) were equally divided among three 31 L tanks (stocking density = 8.6 g L-1) and categorized into three separate groups: unexposed fish infected with lice, A. salmonicida-infected fish infected with lice and reference fish. Fish and sea lice were handled in the same manner as stated in the methods section of the manuscript, except the concentration of A. salmonicida (4.5 x 107 cfu ml-1) was different. At 24 h and 72 h post-infection, three fish from each group were sampled. Salmon skin plugs (5 mm2, methods by Braden et al., 2012) consisting of skin and underlying tissues were immediately removed from sites of louse attachment on each fish and from two similar sites on each reference fish. Samples were immediately pooled via group category, snap frozen in liquid nitrogen and stored at -80oC for subsequent histology. Standard histological methods, using an H & E stain, were performed by L. Braden. Fish infected with lice demonstrated a localized inflammatory response exhibited by epidermal erosion, hyperplasia and ulcerations in the epidermis due to sea lice feeding with cellular aggregation occurring beneath the site (Fig. B.1). Consequently, fish that were infected with A. salmonicida and lice exhibited a more widespread inflammatory response, consisting of a breakdown in tissue integrity, ulcerations, hyperplasia, spongiosis, hemorrhages, erosion and the presence of aggregated neutrophils (Fig. B.2).      68    Figure B.1 Atlantic Salmon (S. salar) Parasitized with L. salmonis. Histological skin samples (5 mm2) taken beneath L. salmonis attachment sites. Localized inflammatory response: a) Epidermal hyperplasia (400x); b) Epidermal erosion (100x); c) Ulceration associated with lice attachment site (400x). (Photos: C. Novak)        a. b. c. 69       Figure B.2 Atlantic Salmon (S. salar) Parasitized with L. salmonis and Infected with A. salmonicida. Histological skin samples (5 mm2) taken beneath L. salmonis attachment sites. Widespread inflammatory response: a) spongiosis (400x); b) haemorrhage (400x); c) tissue degradation (400x); d) neutrophil aggregation (1000x oil); e) coccobacillus-shaped bacteria between epidermal and dermal layers (1000x oil). (Photos: C. Novak)   a. b. c. d. e. 70  Appendix C: Serendipitous Discoveries    Figure C.1 Kudoa thysites Infections in Three Reference Fish from Trial 2. a) Post mortem tissue degradation at 24 h. b) Stellate-shaped spores with four polar capsules (1000x oil); c) Pseudocysts containing hundreds of individual spores in muscle tissue (400x); d) Developing spore stages within pseudocyst (1000x oil). (Photos: C. Novak)        a. b. c. d. 71    Figure C.2 Antithamnion spp. (Rhodophyta) Growing on the Carapace of L. salmonis. a) Several algae filaments on an individual louse. b) Site of algal holdfast on dorsal carapace of sea louse (400x). (Photos: C. Novak)  

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