Evaluation of Rainbow Trout as a Model for use in Studies on Pathogenesis of the Branchial Microsporidian Loma salmonae DAVID J. SPEARE, DVM, DVSC, GARTH J. ARSENAULT, ENV TECH, AND MELANIE A. BUOTE, BS Abstract _ Loma salmonae is an economically important gill microsporidian pathogen of pen-reared chinook and coho salmon. Chinook and coho salmon are generally poorly suited for use in laboratory studies because of their high mortality rates when infected with L. salmonae and their high-level of susceptibility to other infectious diseases. Using gill tissue from chinook salmon that contained mature xenomas laden with L. salmonae spores, we successfully transmitted the infection to rainbow trout. The infection developed in an identical manner and over a similar time course in trout as for chinook salmon. In contrast, we were unable to transmit the infection to other candidate salmonid species, including Atlantic salmon, brook trout, or arctic charr. Gill tissue from experimentally infected rainbow trout was then used to successfully transmit the parasite to other trout. Horizontal transmission was documented from infected to naive tankmates. Analysis of these results indicated that L. salmonae can have a complete life cycle in trout and produce viable spores. Although abundant xenomas developed in the gills of infected trout, the fish did not have clinical signs and there were no fatalities. We concluded that use of rainbow trout offers several key advantages for study of the pathobiologic characteristics of L. salmonae. Gill disease associated with the microsporidian parasite Loma mission of the organism, gill material from infected chinook salmonae is an emerging problem affecting the marine aquacul- salmon was obtained from another research worker (Dr. M. L. ture production of Pacific salmon, most notably chinook salmon Kent, Department of Fisheries and Oceans, Pacific Biological (Oncorhynchus tshawytscha) and, to a lesser extent, coho salmon Station, Nanaimo, British Columbia, Canada ). Gill arches from (O. kisutch) (1–6). Parasites develop in the vasculature of the naturally or experimentally infected fish were removed, placed gills to form large spore-laden xenomas (2–6). Loma salmonae in Earl’s balanced salt solution, and transported on ice. Material also infect rainbow trout (O. mykiss) (7–9); however, in contrast was kept chilled and used 3 days after harvest. to the Pacific salmon, the clinical effects on these fish vary con- In subsequent studies, spores were obtained from gills of in- siderably (9). Furthermore, on the basis of unpublished fected rainbow trout. Spores were obtained 7 to 8 weeks after observations from diagnostic materials sent to the Fish Health fish were exposed to spores during the previous (initial) study. Unit of the Atlantic Veterinary College, L. salmonae is often de- Fish were housed in water at a temperature of 14.58C. Fish with tected in groups of trout that do not have a history of clinical abundant xenomas were selected, euthanatized with an overdose disease, whereas this is not the clinical history for chinook salmon. of benzocaine dissolved in water (100 mg of benzocaine/L of Because of the restrictive nature of current regulations per- water), gill arches were removed, and tissues were used 1 to 3 h taining to the use of therapeutic agents in aquaculture, licenced after collection. therapeutic agents are not available for treating fish infected Fish: All procedures involving live fish were performed accord- with L. salmonae. Accordingly, there is a need to study the ing to guidelines of the Canadian Council on Animal Care. pathobiologic characteristics of L. salmonae in an effort to un- Specific protocols were reviewed by the institutional animal care cover management factors that could limit the economic effects committee (University of Prince Edward Island, Charlottetown, of infection. Laboratory studies of this disease would benefit by Prince Edward Island, Canada). the availability of a species other than chinook salmon, even Juvenile rainbow trout (mean weight, 4 g) were obtained from though they are the principal aquaculture species affected by L. a certified disease-free (specific pathogens) commercial hatch- salmonae. Chinook salmon have proven to be difficult to work ery on Prince Edward Island which did not have a history of L. with in laboratory studies (10) and often become diseased or salmonae infection. Fifteen fish were arbitrarily selected from die because of a range of other endemic infectious diseases, par- 1,000 received and were examined histologically to rule out L. ticularly when the fish are stressed. Rainbow trout, in contrast, salmonae or other diseases not included in federal certification are a relatively hardy fish that are frequently used in laboratory procedures. During the studies, fish weighed from 4 to > 147 g. studies (11). They have been the salmonid species used as a Variations in weight were due to growth, because the various model for many studies, and their requirements and physiologic studies were conducted sequentially. One hundred 3-g Atlantic responses are well documented (11). salmon (Salmo salar) and 3-g brook trout (Salvelinus fontinalis) Our purpose was to evaluate the suitability of rainbow trout as were also acquired from the hatchery where we purchased the an animal model for L. salmonae infections with particular em- trout. One hundred 6-g Arctic charr (Salvelinus alpinus) were phasis on the ability of this parasite to complete its life cycle in a obtained from another commercial hatchery, also with certified manner similar to that seen in naturally and experimentally in- disease-free status. Samples of these other species were exam- fected chinook salmon. ined 4 weeks after their arrival to detect any evidence of L. Materials and Methods salmonae or L. fontinalis. Methods of infection: Fish were exposed to infected gill mate- Source of Loma salmonae spores: In the initial studies on trans- rial by two ways. A feeding method was devised on the basis of Department of Pathology & Microbiology, Atlantic Veterinary College, University previously published methods (6). Approximately 2 g of gill tis- of Prince Edward Island, Charlottetown, Prince Edward Island, Canada C1A 4P3 sue was minced into pieces approximately 0.2 cm2. This tissue 55 was then fed to 20 to 30 fish in a tank. An intubation method was native salmonid species (two non-Oncorhynchus genera). Using devised on the basis of information gained during personal com- spores derived from infected trout, we intubated 20 Atlantic munications (12). Filament cartilage was dissected from salmon, 20 brook trout, and 20 arctic charr. Twenty naive rain- harvested gills, which were then manually crushed. Fish were bow trout were also intubated and used as control fish. Infection manually restrained or lightly anesthetized in a solution of ben- status of fish was monitored weekly through 9 weeks. At week 7, zocaine (40 mg of benzocaine/L of water), and 0.1 to 0.2 ml of samples were collected for histologic assessment to determine the gill tissue-diluent was administered, using gastric intubation. whether xenomas or other evidence of disease were evident in Water supply, housing and characteristics: On arrival, fish were nonbranchial tissues in these test species. During this study, fish stocked in 70-L (salmon, brook trout, arctic charr) or 500-L fi- were housed for 14 weeks in a single 70-L tank supplied with berglass tanks (trout) that received water pumped from a well at freshwater at a temperature of 14.58C. 108C. Fish were fed a recommended amount (13) of a commer- Study 4. To assess whether the final number of xenomas that cially available diet until they entered a study. All studies were developed on the gills of infected fish was correlated with the conducted in 70-L circular tanks supplied with freshwater (2.7 infective dose received, we infected 75 rainbow trout via intuba- L/ min). Dissolved oxygen concentrations were maintained be- tion with 3 dilutions (1:1, 1:10, 1:100) of a preparation of spores. tween 8 and 11 mg/L. For studies conducted with the water A fourth group of 75 fish was intubated with water only. The 1:1 temperature of 14.58C, water from the well was heated and mixed dilution yielded a dose of approximately 4.8 X 106 spores/fish, in a header tank with ambient-temperature water until 14.58C determined on the basis of calculations derived from enumerat- was reached. To avoid supersaturation problems, heated water ing the number of xenomas used and a volumetric calculation was degassed through a trickle column before use. of the reported theoretical maximum number of spores per Evaluating infection status: For studies conducted by using xenoma (8). Seven weeks after intubation, fish were euthana- water at a temperature of 14.58C, the infection status of fish was tized by immersion in an overdose of benzocaine. The second assessed two or more times, usually during the period 5 to 7 gill arch on the left side of each fish was removed, and number weeks after initial exposure to infective material. For studies con- of xenomas was counted, using a compound stereomicroscope. ducted by using water at colder temperatures or when species Number of gill filaments on the arch was also counted. Data other than rainbow trout were used, more-frequent screening were reported as number of xenomas per filament to compen- during a longer period was used. During each assessment, fish sate for any differences in gill surface area that may have resulted were anesthetized in benzocaine (60 mg of benzocaine/L of from potential differential growth rates between groups of fish water). The operculum was lifted, and the gills were examined, exposed to differing doses. using a dissecting microscope. The entire surface of the second Effect of dilution on number of xenomas formed was assessed gill arch of each fish was examined for evidence of xenomas.