Transmission of salmonae (Microsporea) to chinook salmon in sea water Michael L. Kent, Sheila C. Dawe, David J. Speare

Abstract poissons devraient considerer cette possibilite lors de Transmission studies were conducted to determine if l'elaboration et de l'implantation de leur plan was transmissible in sea water. strategique pour controler les infections. Transmission of L. salmonae to chinook salmon (Traduit par Dr Therase Lanthier) ( tshawytscha) held in sea water was achieved by exposing fish to macerated, infected gill Can VetJ 1995; 36: 98-101 tissue. Fish were exposed in seawater in a flow- through aquarium, and the infection was detected as Introduction soon as 5 wk after exposure. Heavily infected fish L oma (formerly Pleistophora) salmonae infects the exhibited numerous xenomas in the branchial arter- gills and other vascularized tissues of wild and ies, central venous sinusoids, and within the blood hatchery-reared salmonids in fresh water throughout channels of the lamellae. The pathological changes the Pacific Northwest (1-7). Severe gill infections were similar to those seen in pen-reared salmon with have been reported in rainbow and steelhead trout L salmonae infections. The infection was not observed (Oncorhynchus mykiss) and kokanee salmon (0. nerka) in Atlantic salmon (Salmo salar), Pacific herring (1). Hauck (4) observed high mortality in chinook (Clupea pallasi, family Clupeidae), or shiner perch salmon (0. tshawytscha) due to systemic infections by (Cymatogaster aggregata, family Embiotocidae), a Loniia sp. (presumably L. salmonae). Although the experimentally exposed using identical methods. gill is the primary site of infection, parasites and asso- This study suggests that L. salmonae is transmissible ciated lesions can occur elsewhere, including the heart, to chinook salmon in seawater netpens. Fish farmers spleen, kidney, and pseudobranch (8). and fish health specialists should consider this pos- Loma salmonae is considered to be a freshwater par- sibility when developing and implementing strategies asite, but infections can persist after fish are trans- to control the infection. ferred to seawater (9). In seawater netpens, severe gill lesions associated with the infection have been observed Resume in coho salmon (0. kisutch) (8,10) and chinook salmon Transmissibilite de Loma Salmonae (Micro- (1 1). Morbidity associated with L. salmonae infections sporea) au saumon chinook en eau salee has increased recently in pen-reared chinook salmon in Une etude a ete effectuee afin de determiner la trans- British Columbia, according to reports from fish farmers missibilite de Loma salmonae au saumon chinook and fish health specialists. garde en eau salee dans un aquarium 'a flux 'a debit Experimental transmission studies with other continu. La transmission de L. salmonae au saumon of fishes indicate that most are directly chinook (Oncorhynchus tshawytscha) a ete accomplie transmitted and do not require intermediate hosts, en exposant le poisson a du tissu macere d'ouies although aquatic crustaceans may act as vectors for infectes. L'infection a ete decelee 5 semaines apres some microsporidia (12). In a previous study of pen- l'exposition. Les poissons fortement infectes demon- reared coho salmon, it was clear that some fish car- traient plusieurs xe'nomes dans les arteres branchiales, ried the infection from fresh water, but it was incon- dans les sinusoides veineux centraux et dans les vais- clusive whether the infection was transmitted from seaux des lamelles. Les changements pathologiques infected to uninfected fish within the seawater pens etaient semblables a ceux observes chez les saumons (8). A high prevalence of the infection was recently infectes gardes dans un bassin. observed in chinook salmon at one netpen farm in L'infection n'a pas ete observee chez le saumon de British Columbia, where all the fish had been reared on, l'Atlantique (Salmo salar), le hareng du Pacifique presumably, L. salmonae-free wellwater during their (Clupea pallasi, famille Clupeidae) et la perchaude freshwater phase of development. This suggested that (Cymatogaster aggregata, famille Embiotocidae) lors many, if not all, of these fish contracted the infection in des essais experimentaux. Cette etude suggere que seawater. L. salmonae est transmissible au saumon chinook The purpose of this study was to determine if garde' en eau salee dans des cuvettes en filet. Les L. salmonae is transmissible to chinook salmon in sea- pisciculteurs et les specialistes des pathologies des water and to describe the pathological changes in the gills due to L. salmonae, in the absence of other concurrent Department of Fisheries and Oceans, Pacific Biological Station, diseases of the gills that are frequently observed in Nanaimo, British Columbia V9R 5K6 (Kent, Dawe) and pen-reared salmon. Atlantic salmon (Salmo salar) is Department of Microbiology and Pathology, Atlantic Veterinary the most important species reared in seawater netpens in College, University of Prince Edward Island, 550 University British Columbia. Shiner perch (Cymatogaster aggre- Avenue, Charlottetown, Prince Edward Island CIA 4P3 gata) and Pacific herring (Clupea pallasi) are the most (Speare). common nonsalmonid wild fishes that enter and inhabit Funded in part by the British Columbia Ministry of Agriculture, these netpens. Therefore, the susceptibility of these Fisheries and Food. fishes to L. salmonae was also investigated. 98 Can Vet J Volume 36, February 1995 Materials and methods The transmission experiments were done at the Pacific Biological Station (PBS), Nanaimo, British Columbia in tanks with flow-through seawater (salinity 28 to 30 parts/ thousand). The seawater for this laboratory is pumped from a depth of approximately 30 m and is processed through sand filters that remove particles > 25 pm. Approximately 5 g of chinook salmon gill tissue containing numerous L. salmonae xenomas (collected from a commercial netpen site) were placed in sterile saline, chopped finely, and introduced to the aquar- ium. The water flow to the tank was turned off for 2 h to enhance contact of the parasite with the fish. Fish were exposed 3 times on alternate at days the beginning of Figure 1. Loma salmonae infection in chinook salmon gill at each experiment. 8 wk postexposure. Arrowheads = intact xenomas. Epithelial At the end of each experiment, fishes were euthanized hyperplasia (E) associated with chronic inflammation and with an overdose of tricaine methanesulfonate eosinophilic material with fibrin (F) in central venous sinusoids. (MS-222, Syndel Laboratories, Vancouver, British Hematoxylin and eosin. Bar = 500 pm. Columbia). Four gill arches were collected from each fish and fixed in Davidson's solution (13). Microsections were prepared from each arch, stained with hematoxylin and and for the (Figure 1). Ten control fish maintained under the same eosin, examined presence of L. salmonae conditions did not and associated lesions. Some sections were also stained exhibit the infection. with modified Russell-Movat stain The general architecture of the gills from heavily pentachrome (14) and infected fish was altered Martius scarlet blue stain (15) to verify the presence of markedly due to multifocal dis- fibrin. tribution of xenomas and associated inflammatory responses in and around blood vessels and blood chan- nels. Numerous xenomas were observed within the Experiment 1 afferent and efferent arteries Twenty chinook salmon smolts (avg wt 22 were in the branchial arch and g) the central venous sinusoids (CVS) of the filaments divided equally into 2 groups of 10 each. Each group was and placed in a 40 L and 1 was (Figures 1 2). Many of the xenomas appeared to have tank, group exposed as a focal attachment to the described. Water temperature was maintained at 1 °C to endothelium, and spores were 15C throughout the study. Both exposed and control fish entirely bound by the margin of the . Many were examined at 5 and 8 wk after the xenomas protruded into the lumen of the vessel, while initial exposure. others extended outward through the tunica adventitia Experiment 2 (Figure 2). Some of the latter extended through the basement membrane of the overlying filament epithelium. Twenty chinook salmon (avg wt 30 g) and 20 Atlantic The salmon (avg wt 80 g) were maintained in a 725 L tank regional distribution of the xenomas favored the dis- tal two-thirds of each filament. The host reaction to and exposed to infected gill tissue. An additional 20 chi- intact nook salmon, 20 Pacific herring (approx avg wt xenomas varied from 1) no detectable response, 10 g) and 10 shiner perch (approx avg wt 20 g) were like- to 2) minimal aggregation of thrombocytes and mono- wise exposed and maintained in another 725 L tank. nuclear cells associated with the intraluminal portion of the xenoma, to 3) features of 2 above, plus a mild peri- Control chinook salmon were maintained in a third vascular 725 L tank. The Atlantic salmon were stock fish that had cuff of mononuclear cells and fibroblasts. been reared on fresh water at the PBS prior to the study, Xenomas were also common within lamellae. whereas the Pacific herring and shiner perch were Although in many cases the structure of the lamella "wild-caught" from Departure Bay, Nanaimo, British was obliterated by xenomas, the xenomas appeared to Columbia. Fishes in the tank containing the herring develop within both pillar cells and the endothelial and perch were also fed chopped frozen euphasid cells of the marginal channel. Fibrin was commonly shrimps daily, because it was noted that the herring found in pillar channels, but little inflammation was did not feed well on the commercial salmon diet. This associated with xenomas in this location. experiment was terminated 7 wk after the initial expo- Approximately 25% of the xenomas in the branchial sure, and all fish from the three tanks were killed and arch and filaments were in stages of degeneration. As in examined by histotogy. previous reports (8,10), degenerated xenomas were associated with severe fibrinoid arteritis, periarteritis, and hyperplasia of the overlying epithelium. In some Results instances, remarkable amounts of eosinophilic material Experiment 1 were present, and this material often obliterated entire At 5 wk postexposure, 4 of 5 exposed chinook salmon lumens of the CVS. This eosinophilic material con- exhibited massive hypertrophic lesions (xenomas) due tained large areas within it that stained red to intensely to L. salmonae within the vasculature of the filaments and red with either modified Russell-Movat pentachrome lamellae. Examination of 4 of the remaining 5 fish at stain or Martius scarlet blue stain, which indicated that it 8 wk postexposure revealed that 3 were infected and 2 contained abundant fibrin (14,15). Free spores were often of them had severe pathological changes in the gills observed within this fibrin accumulation (Figure 2).

^-- 't I -L I t 1- 1. %- r- .- I --- Can Vet J Volume 36, February 1995 99 (16) experimentally transmitted atherinae of sand smelt (Atherina presbyter) to turbot (Scophthalmus maximnus), and Wales and Wolf (1) reported an infection of L. salmonae in a freshwater sculpin (Cottus sp.). However, shiner perch and Pacific herring (the most com- mon nonsalmonid fishes found in netpens in British Columbia) were both resistant to the infection. Atlantic salmon were also resistant to the infection. However, these results should be considered preliminary, because the Atlantic salmon were much larger than the chi- nook, and perhaps the size and the age of salmon affected their susceptibility. Gill tissue deteriorates rapidly after death. Therefore, t ~ ~ ,,/- ,.1 *'-', 1s X_ tissue xenomas Figure 2. Intact xenomas (X) occluding lumen of central fragments containing and spores would venous sinusoid and extending through the tunica adventitia. be released into the water shortly after the death of an Free spores (S) and eosinophilic exudate with fibrin (F) in CVS. infected fish. Many netpen farmers leave dead fish in Hematoxylin and eosin. Bar = 50 ,um. pens for several days, and these carcasses are probably a source of infection to other salmon in the pen. Fish farmers should consider removing L. salmonae-infected 2 Experiment carcasses as soon as possible, to minimize contact of All fish species ate pieces of infected tissue intro- gill uninfected fish with the parasite. Dead fish are usu- duced to the tank. the Pacific fed However, herring ally removed by scuba divers, who collect the carcasses poorly on the commercial diet, and during the I st 3 wk into net bags and hoist the dead fish through the water of the study, 13 herring became very emaciated and column within the netpen. During this operation, a con- died. Therefore, the chinook salmon were removed siderable amount of tissue debris is released into the from the tank containing perch and because herring, water column, and fish feed on this material. Because this their aggressive feeding behavior inhibited the herring practice could further enhance transmission of from receiving adequate food, and were in the placed L. salmonae and other pathogens, we recommend that 725 L tank containing the exposed Atlantic salmon. this operation be modified (e.g. remove carcasses in Three chinook died during the study, but were not watertight bags). The farm staff cooperating with us examined. in this study has already used this method without an Seven weeks after exposure, 19 of the remaining unreasonable increase in time and effort. 37 exposed chinook exhibited infection with L. salmonae. The gill lesions in the fish at 8 wk postexposure were In all xenomas were infected chinook, intact and there similar to those observed in coho salmon with was very little was associated inflammation. Infection not L. salmonae infections from netpens (8,10). The tissue detected in any of the Atlantic salmon, Pacific herring reaction was also consistent with the response generally (5 surviving fish), shiner perch, or 20 control chinook. associated with xenoma-forming microsporidia; that is, inflammatory changes are most severe when xenomas Discussion begin to degenerate and liberate spores (17). Kent et al Our ability to transmit L. salmonae directly with fresh (8) suggested that degenerating xenomas in lamellae spores is not surprising, given that several other fish released spores directly into the water and thus were asso- microsporidia are transmitted in this manner. However, ciated with little inflammatory reaction. In contrast, the transmissibility of this "freshwater" parasite in sea- spores released into gill tissue remain until they are water has significant implications for chinook salmon net- eliminated by the active tissue response that they elicit. pen farming. For example, a low prevalence of infection This probably accounts for the severe inflammatory in a population of smolts at the time of seawater trans- changes in the CVS and branchial arteries. fer would mean that a future epizootic could occur in this The presence of perivascular eosinophilic material con- population due to fish-to-fish transmission within the pen. taining fibrin indicates vessel damage and leakage of Furthermore, chinook salmon in netpens could become blood proteins. The formation of fibrin reflects endothe- infected from other populations of infected chinook lial damage and activation of coagulation Factor XII salmon at the farm or from wild salmonids in the vicin- (Hageman). This is consistent with earlier findings in nat- ity. According to reports from fish farmers at the affected urally occurring L. salmonae infections in which a netpen site used in this study, several wild chum salmon leukocytoclastic vasculitis with fibrinoid necrosis of (0. keta) entered the pens as juveniles and resided in the the tunica media was described (10). Fibrinoid vas- pens for several months. Therefore, the chum salmon culitis is also a feature of Encephalitozoon cuniculi may have been the source of the infections, although infections in mammals (12,18). L. salmonae has not been reported from this salmon In conclusion, the ability to transmit L. salmonae in species. the laboratory and to recreate pathological changes The possibility of a nonsalmonid marine fish reservoir similar to those observed in naturally-infected fish will for L. salmonae should also be considered. Although facilitate research on the pathogenesis and host specificity most microsporidia have a narrow host specificity, of the parasite. Furthermore, the efficacy of potential some can infect a wide range of host species (e.g. chemotherapeutics to control the infection can be stud- Encephalitozoon cuniculi of mammals) (12). Leiro et al ied in a controlled, laboratory setting. cvI

100 Can Vet J Volume 36, February 1995 References 10. Speare DJ, Brackett J, Ferguson HW. Sequential pathology of the gills of coho salmon with a combined diatom and microsporidian 1. Wales JH, Wolf H. Three protozoan diseases of trout in California. gill infection. Can Vet J 1989; 30: 571-575. Calif Fish Game 1955; 41: 183-187. 11. Kent ML. Diseases of seawater netpen-reared salmonid fishes in 2. Putz RE, Hoffman GL, Dunbar CE. Two new species of the Pacific Northwest. Canadian Special Publication of Fisheries Plistophora (Microsporidea) from North American fish with a syn- and Aquatic Sciences 116; 1992: 39-42. opsis of Microsporidea of freshwater and euryhaline fishes. 12. Canning EU, Lom J. The Microsporidia of Vertebrates. Toronto, J Protozool 1965; 12: 228-236. Academic Press, 1986. 3. Putz RE, McLaughlin JJA. The biology of Nosematidae 13. Humason GL. Animal Tissue Techniques, 4th ed. San Francisco: (Microsporida) from freshwater and euryhaline fishes. In: Snieszko WH Freeman, 1979: 470. SF, ed. A Symposium on Diseases of Fishes and Shellfishes. 14. McElroy DA. Connective tissue. In: Prophet EB, Mills B, Arrington Special Publ No. 5, Am Fish Soc., Washington, DC, 1970: JB, Sobin LH, eds. Armed Forces Institute of Pathology, Laboratory 124-132. Methods in Histotechnology. Washington, DC: American Registry 4. Hauck AK. A mortality and associated tissue reactions of chinook of Pathology, 1992: 127-129. salmon, Oncorhynchus tshawytscha (Walbaum), caused by the 15. Drury RAB, Wallington EA. Carleton's Histological Technique, microsporidan Loma sp. J Fish Dis 1984; 7: 217-229. 5th ed. Oxford: Oxford University Press, 1980: 229. 5. Morrison CM, Sprague V. Microsporidian parasites in the gills of 16. Leiro J, Bos R, Iglesias J, Esteves J, Fernandez J, Sanmartin salmonid fishes. J Fish Dis 1981; 4: 371-386. ML. Experimental infection of turbot (Scophthalmus maximus L.) 6. Morrison CM, Sprague V. Loma salmonae (Putz, Hoffman and with a microsporean parasite (Glugea atherinae Berrebi 1979) of Dunbar, 1965) in the rainbow trout, Salmo gairdneri Richardson, the sand smelt (Atherina presbyter C.). Aquaculture 1993; 118: 1-7. and L. fontinalis sp. nov. (Microsporida) in the brook trout, 17. Dykova I, Lom J. Tissue reaction to microsporidian infections in Salvelinusfontinalis (Mitchell). J Fish Dis 1983; 6: 345-353. fish. J Fish Dis 1980; 3: 265-283. 7. Magor BG. First report of Loma sp. (Microsporidia) in juvenile 18. Jubb KVF, Huxtable CR. In: Jubb KVF, Kennedy PC, Palmer N, coho salmon (Oncorhynchus kisutch) from Vancouver Island, British Columbia. Can J Zool 1987; 65: 751-752. eds. Pathology of Domestic Animals, 4th ed. Vol 1. Toronto: 8. Kent ML, Elliot DG, Groff JM, Hedrick RP. Loma salmonae Academic Press, 1993: 267-437. (Protozoa: Microspora) infections in seawater reared coho salmon Oncorhynchus kisutch. Aquaculture 1989; 80: 211-222. 9. Wood JW. Diseases of Pacific Salmon, Their Prevention and Treatment. Olympia, Washington: State of Washington, Department of Fisheries, 1974: 42. Available from Department of Fisheries, 115 General Administration Building, Olympia, Washington 98504, USA.

Continuing \ Edu(ation \ Program:Fline& T Canine Behaviour Speaker-Dr. Wayne LAKE LOUISE, ALBERTA Hunthausen, pet behaviour 5 consultant and practitioner from Kansas City. Sessions will be moderated by /Y / FEBRUARY 22-25, 1995 Dr. Gary Landsberg, pet behaviour consultant I and practitioner from Toronto. CHATEAU LAKE LOUISE

Full package for $888.00 (CVMA members) includes: nine hours of continuing education, refreshment breaks, welcome Bring your kis... and cocktails, 4 nights accommodation at the beautiful Chateau Lake your behaviour cases Louise, 3 hearty breakfasts, and 3 days of skiing. Packages will be available for accompanying persons, and cross-country skiers. Registration is limited so book earlv! m iF Deadline to register is January 10, 1995. For more information, contact Carol Bell, CVMA Iffij 1-800-567-2862 (in 0~This conference is co-sponsored by Veterinary Medical Diets Inc. Canada) or (613) 236-1162 Can Vet J Volume 36, February 1995 101