1996 rickettsial infection in fish. fryer

Pergamon Annual Review of Fish Diseases, Vol. 6, pp. 3-13, 1996

Copyright 0 1997 Published by Elsevier Science Ltd Printed in Great Britain. All rights reserved

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PII:SO959-8030(96)00001-4

RICKETTSIAL INFECTIONS OF FISH*

J. L. Fryer? and C. N. Lannan Department of Microbiology, Oregon State University, Nash Hall 220, Corvallis, OR 97331-3804, USA

Absrract. Pisciricketfsia salmonis is the first of the previously unrecognized rickettsial pathogens of fish to be isolated, characterized, and demonstrated to be the etiologic agent of an epizootic disease. However, since the isolation of P salmonis in 1989. the scope and impact of these pathogens has become increasingly apparent. This growing awareness of the role of the rickettsiae in fish health has led to documentation of rickettsial diseases in diverse species of fish and in disparate geographic locations and aquatic environments. Considerable work remains in order to establish the source, reservoir, and normal mode(s) of transmission of these agents and to devise appropriate methods of disease prevention and control. 0 1997 Published by Elsevier Science Ltd

Krywords: Rickettsia, Fish disease, Piscirickertsia salmonis, Piscirickettsiosis

INTRODUCTION

Aquatic animals are host to a wide variety of intracellular rickettsia-like and chlamydia-like Gram negative bacteria (1). Of these, only the diverse group of chlamydia-like organisms responsible for epitheliocystis, although never cultured or characterized, have traditionally been considered pathogens of fish. However, in recent times, this limited view of the role of intracellular bacteria in fish disease has dramatically changed, and rickettsial agents have assumed an important place among those bacterial pathogens with significant impact on fish health.

HISTORY

The role of the rickettsiae in fish disease became apparent in 1989 (2), when a previously unrecognized rickettsia [Piscirickettsia salmonis gen. nov., sp. nov., (3)] was isolated in cultures of the chinook salmon embryo cell line, CHSE-214 (4), and demonstrated to be the cause of devastating epizootics in marine-netpen-reared coho salmon Oncorhynchus kisutch in southern Chile (5). It was originally thought that the infections were specific for coho salmon, but subsequently, piscirickettsiosis was also observed in the other salmonid species cultured in the area [chinook salmon Oncorhynchus tshawyfscha, Atlantic salmon Salmo salar, rainbow trout Oncorhynchus mykiss , and masu salmon Oncorhynchus masou; (6)].

Piscirickettsia salmonis was the first rickettsia of aquatic poikilotherms to be isolated, characterized, and demonstrated to produce disease in fish. On two earlier occasions, rickettsia- like agents were observed in non-salmonid fish species at diverse locations and in differing aquatic environments around the world (7,8) and one such agent from rainbow trout was isolated in cell culture (9), but none of these organisms was identified or characterized and their role in the fish disease process remains unknown.

Since 1989, a growing number of reports have documented rickettsial diseases in fish (Table 1). Currently, there are at least 50 publications that detail one or more aspects of the infection of

*Oregon Agricultural Experiment Station Technical Paper 10,846 tTo whom correspondence should be addressed.

3

4 J.L. Fryer and C.N. Lannan

Table I. Rickettsial and rickettsia-like organisms observed in or isolated from fish

Host species Geographic Region Salt/Fresh Water Isolated/Observed Reference

Tetrodon fahaka Rainbow trout

(Oncorhynchus mykiss) Dragonet

(Callionymus lyra) Coho salmon

(Oncorhynchus kisutch) Rainbow trout Chinook salmon

(Oncorhynchus rshawyrscha) Atlantic salmon

(Salmo salar) Chinook salmon Atlantic salmon Coho salmon Pink salmon

(Oncorhynchus gorbuscha) Atlantic salmon Atlantic salmon Sakura salmon

(Oncorhynchus masou) Tilapia spp. Blue-eyed plecostomus

(Panaque surtoni) Coho salmon Rainbow trout Atlantic salmon Tilapia nilotica x Oreochromis nilotica Sea bass

(Dicenrrarchus labrax)

Egypt Europe

Wales

Chile

Chile

Pacific Coast Canada

Ireland Norway

Chile

Taiwan Colombia

Chile

Chile Japan

France

F 0 (7) F Id (9)

Sb 0 03)

S I* (2)

s o* (5)

S O- (29)

S 0* (12) S oI* (11) S 0 (6)

S&F 1 (13) F 0 (14)

F 1* (16)

F I (17) F 0 (30)

S 0 (15)

a F = fresh water; b S = salt water; c 0 = observed; d I = isolated; e = subsequently isolated; * = identified as Piscirickettsia salmonis.

fish by rickettsial or rickettsia-like organisms (Tables 2 and 3). Of these, fewer than half appear in the peer-reviewed literature (Table 2). However, an increasing number of scientific papers concerning rickettsial infections in fish are published in journals subject to peer review, and at present, at least 20 peer-reviewed papers on this topic can be found. We believe the trend toward formal documentation of the role of the rickettsiae in fish health will and should increase.

Following the identification of I! sulmonis in Chilean salmonids (2) similar infections were identified in salmonid fish at three locations in the northern hemisphere. In the western Canadian province of British Columbia, Brocklebank er al. (10) found a disease in maricultured Atlantic salmon that resulted in pathology indistinguishable from that observed in the salmonids in Chile. This disease was caused by an organism morphologically identical to F! salmonis, but unlike the P safmonis infections in Chile, it produced only low level mortality in the infected fish. These researchers also reported that an analogous disease occurred, as early as 1970, in pink salmon Oncorhynchus gorbuscha, held in seawater tanks in British Columbia, and in coho and chinook salmon seafarmed there in the 1980s. Rickettsiae morphologically similar to those implicated in the diseases in Chile and in Canada also caused low level mortality in Atlantic salmon held in

Rickettsial infections of fish 5

Table 2. Reports on rickettsial and rickettsia-like pathogens of fish: The peer-reviewed literature

Alday-Sanz, V., Rodger. H., Turnbull, T.. Adams, A., Richards, R.H. (1994). An immunohistochemical diagnostic test for rickettsial disease. J. Fish Dis. 17:189-191.

Branson, E.J., Nieto Diaz-Munoz, D. (1991). Description of a new disease condition occurring in farmed coho salmon, Oncorhynchus kisutch (Walbaum), in South America. J. Fish Dis. 14:147-156.

Brocklebank, J.R., Speare, D.J., Armstrong, R.D., Evelyn T. (1992). Septicemia suspected to be caused by a rickettsia- like agent in farmed Atlantic salmon. Can. Vet. J. 33:407-408.

Chen, S.-C., Tung, M.-C., Chen, S.-P., Tsai, J.-F., Wang, P.-C., Chen, R.-S., Lin, S.-C., Adams, A. (1994). systemic granulomas caused by a rickettsia-like organism in Nile tilapia, Oreochronuis niloticus (L.), from southern Taiwan. J. Fish Dis. 17591-599.

Chem, R.S., Chao, C.B. (1994). Outbreaks of a disease caused by a rickettsia-like organism in cultured tilapias in Taiwan. Fish Pathol. 29(2):61-71.

Cvitanich, J.D., Garate N., 0.. Smith, C.E. (1991). The isolation of a rickettsia-like organism causing disease and mortality in Chilean salmonids and its confirmation by Kochs postulate. J. Fish Dis. l4:12l-145.

Fryer, J.L., Lannan, C.N. (1992). Isolation of a rickettsia from diseased salmonids. In: Kimura, T. (ed.) Salmonid Diseases. Hokkaido University Press, Hakodate, pp. 151-157.

Fryer, J.L., Lannan, C.N. (1994) Rickettsial and chlamydial infections of freshwater and marine fishes, bivalves, and crustaceans. Zool. Studies 33(2):95-107. [Translated into Japanese by Professor Tokuo Sano and appears in Fish. Res. (1995) 14(3):54--65.]

Fryer, J.L., Lannan, C.N., Garcbs, L.H., Latenas, J.J., Smith, P.A. (1990) Isolation of a rickettsiales-like organism from diseased coho salmon Oncorhynchus kisutch in Chile. Fish Pathol. 25(2):107-l 14.

Fryer, J.L., Lannan. C.N., Giovannoni, S.J., Wood, N.D. (1992). Piscirickettsia salmonis gen. nov., sp. nov., the causative agent of an epizootic disease in salmonid fishes. Int. J. Syst. Bactetiol. 42(1):120--126.

Gaggero, A., Castro, H., Sandino, A.M. (1995) First isolation of Piscirickettsiu salmonis from coho salmon, Oncorhynchus kisutch (Walbaum), and rainbow trout, Oncorhynchus mykiss (Walbaum), during the freshwater stage of their life cycle. J. Fish Dis. l&277-279.

Games, L.H., Larenas, J.J., Smith, P.A., Sandino. S., Lannan, C.N., Fryer, J.L. (1991). Infectivity of a rickettsia isolated from coho salmon (Oncorhynchus kisutch). Dis. aquat. Org. l1:93-97.

Khoo, L., Dennis, P.M., Lewbart, G.A. (1995). Rickettsia-like organisms in the blue-eyed plecostomus, Pannque suttoni (Eigenmann & Eigenmann). J. Fish Dis. 18:157-164.

Lannan, C.N., Ewing, S.A., Fryer, J.L. (1991). A fluorescent antibody test for detection of the rickettsia causing disease in Chilean salmonids. J. Aquat. Animal Hlth 3:229-234.

Lannan. C.N., Fryer, J.L. (1993). Piscirickettsio sohnonis, a major pathogen of salmonid fish in Chile. Fish. Res. 17:115-121.

Lannan, C.N., Fryer, J.L. (1994). Extracellular survival of Piscirickettsia salmonis. J. Fish Dis. 17:545-548. Larenas H., J., Hidalgo V., L., Games A., H., Fryer, J.L., Smith S., P. (1995). Piscirickettsiosis: Lesiones en

salm6n del Atlarttico (S&no s&r) infectados naturahnente con Piscirickettsia salmonis. (In Spanish with English abstract) Avarices en Ciencias Veterinatias 10(1):53-58.

Mauel. M. J.. Giovannoni, S.J., Fryer, J.L. (1996). Development of polymerase chain reaction assays for detection, identification, and differentiation of Piscirickettsia salmonis. Dis. aquat. Org. 26: 189-195.

Ozel, M., Schwartz-Pfitzner, 1. (1975). Vergleichende elektronenmikroskopische Untersuchungen an Rhabdoviren pflanzlicher und tierischer Herkunft: 111. Egtved-Virus (VHS) der Regenbogenforelle (Salmo gairdneri) und Rickettsienahnliche Organismen. Zbl. Bakt. Hyg., 1. Abt Orig. 230:1-14.

Rodger, H.D., Drinan, E.M. (1993). Observation of a rickettsia-like organism in Atlantic salmon, S&to solar L.. in Ireland. J. Fish Dis. 16:361-369.

seawater cages in Norway (11) and in Ireland (12). All of these agents were identified as F! salmonis by fluorescent antibody tests using a polyclonal antiserum.

Until 1994, all documented rickettsial diseases of fish occurred in salmonids cultured in sea water. However, it is now apparent that these pathogens affect fish over a broad host and geographic range and in both freshwater and marine environments. Chern and Chao (13) found an epizootic rickettsial disease in several species of tilapia (Oreochromis mossambicus, Oreochromis niloticus, Oreochromis aureus, Wapia zillii, and Tilapia hornorum) in both freshwater and seawater ponds in Taiwan. Like the ongoing epizootics in Chilean salmonid fish,


Table 3. Reports on tickettsial and rickettsia-like pathogens of fish: The non-peer-reviewed Literature

Anonymous (1991). Animal Viruses. Quart. Newsletter. 11(3):5. Anonymous (1995). Piscirickettsiosis. In: OIE Diagnostic Manual for Aquatic Animal Diseases, 1st edition. Office/

American Type Culture Collection International des Epizooties Paris, France, pp. 135-140. Bravo, S. (1994). First report of Piscitickeftsia salmonis in freshwater. FHSlAFS Newsletter 22( 1):3. Bravo, S. (1994). Piscirickettsiosis in freshwater. Bull. Eur. Assoc. Fish. Pathol. 14(4):137-138. Bravo, S., Campos, M. (1989). Coho salmon syndrome in Chile. FHS/AFS Newsletter 17(3):3. Brocklebank, J.R., Speare, D.J., Armstrong, R.D., Evelyn, T. (1992). Septicemia in farmed Atlantic and chinook

salmon due to a rickettsia-like agent. FHSlAFS Newsletter 20(l): 1. Comps, M., Raymond, J.C., Plassiart, G.N. (1996). Rickettsia-like organism infecting juvenile sea-bass

Dicentrarchus labrax. Bull. Eur. Assoc. Fish. Pathol. 16(1):3&33. Cubillos, V., Far& C., Alberdi, A., Alvarado, V., Schafer, W., Monras, M. (1990).Caractetisticas

anatomopatolbgicas de1 sindrome de1 salmon coho (SSC), nueva enfermedad de 10s salmonfdeos. Patologfa Animal 4:14-17.

Cubillos, V.. Pamdes, E., Monras, M., Alberdi, A. (1995) Salmonid rickettsial syndrome agent determination (SRSAJA) in wild fishes from the province of Palena, Tenth Region (Chile). (Abstract) Eur. Assoc. Fish Pathol. 7th Intemat. Conf. Diseases of Fish and Shellfish, Sept. 10-15. 1995, Palma de Mallorca, Spain

Cvitanich, J.D., Garate N., 0.. Silva P., C., Andrade V., M., Figueroa P., C., Smith, C.E. (1995). Isolation of a new rickettsia-like organism from Atlantic salmon in Chile. FHS/AFS Newsletter 23(3):1-3.

Cvitanich, J.D., &rate N.,O., Smith, C.E. (1990). Etiological agent in a Chilean coho disease isolated and confirmed by Kochs postulates. FHS/AFS Newsletter 18(1):1-2.

Cvitanich, J.D., Garate N.,O.. Smith, C.E. (1995). Isolation of a new rickettsia-like organism from Atlantic salmon in Chile. (Abstract) Western Fish Disease Workshop, June 6-9, 1995, Moscow, ID.

Davies, A.J. (1986). A rickettsia-like organism from dragonets, Callionymus lyra L. (Teleostei: Callionymidae) in Wales. Bull. Eur. Assoc. Fish Pathol. 6(4):103.

Enriquez, R. (1995). Actual situation of the disease produced by Piscirickerrsia salmonis in Chile. (Abstract) Eur. Assoc. Fish Pathol. 7th Intemat. Conf. Diseases of Fish and Shellfish, Sept. lC-15, 1995, Palma de Mallorca, Spain

Evelyn, T.P.T. (1992). Salmonid rickettsial septicemia. In: Kent, M.L. (ed.) Diseases of Seawater Netpen-reared Salmonid Fishes in the Pacific Northwest. Can. Spec. Pub. Fish. Aquat. Sci. 116. Dept. Fisheries and Oceans, Nanaimo, B.C., pp.18-19.

Fryer, J.L., Lannan, C.N. (1990). Preliminary characterization of a rickettsia-like pathogen of salmonids. (Abstmct) In: VIII* International Congress of Virology. Intemat. Union Microbial. Sot., Berlin, p. 49.

Fryer, J.L., Lannan, C.N. (1996). The emergence of rickettsia as important pathogens of fish. (Abstract) American Society for Rickettsiology and Rickettsial Diseases 12th Sesqui-Annual Meeting, May 10-13, 1996, Pacific Grove, CA.

Fryer, J.L., Lannan. C.N., Garc&., L.H., Larenas, J.J., Smith, P.A. (1991). Piscirickerrsia salmonis, a newly described intracellular pathogen of salmonid fish. In: Short Communications of the 1991 International Marine Biotechnology Conference (IMBC 91). Developments in Industrial Microbiology Extended Abstract Series Vol. II. Wm. C. Brown Pub., Dubuque, lA, pp.567-572.

Gaggero. A. (1995). Evaluation of the in vitro sensitivity of Piscirickettsia salmonis to diffetent antimicrobial compounds. (Abstract) Eur. Assoc. Fish Pathol. 7th Intemat. Conf. Diseases of Fish and Shellfish, Sept. IO- 15, 1995, Palma de Mallorca, Spain

Garces, L.H.. Cormal, P., Larenas, J.J., Contreras, J.. Oyanedel, S., Fryer, J.L., Smith S., P. (1995). Finding of Pisciricketrsia salmonis on Cerarhorhoa gaudichaudii. (Abstract) Int. Symp. Aquat. Animal Health, Sept.4-8, 1994. Seattle, WA.

Lannan, C.N., Cvitanich, J.D., Evelyn, T.P.T. (1992). Rickettsial disease of salmonids. Pacific Northwest Fish Health Protection Committee Informational Report 6, 1 lpp.

Lannan, C.N., Fryer, J.L. (1991). Recommended methods for inspection of fish for the salmonid rickettsia. Bull. Eur. Assoc. Fish Pathol. 11:135-136.

Lamnas, J.J., Smith, P.A., Gar&, L.H., Lannan, C.N., Fryer, J.L. (1995). Piscitickettsiosis: Histopathology of Atlantic salmon (Salmo salar) and coho salmon (Oncorhynchus kisurch) inoculated with P. salmonis. (Ab- stract) Int. Symp. Aquat. Animal Health, Sept+8, 1994. Seattle, WA.

Mauel. M.J., Fryer, J.L. (1996). Evidence of genetic diversity within Piscirickertsia salmonis. (Abstract) American Society for Rickettsiology and Rickettsial Diseases 12th Sesqui-Annual Meeting, May 10-13, 1996, Pacific Grove, CA.

Mohamed, 2. (1939). The discovery of a rickettsia in a fish. Minist. Agric. Cairo, Tech. Sci. Serv., Vet. Sect. Bull. 214. 6pp.

Olsen, A.B., Evensen. 0.. Speilberg, L., Melby, H.P. Hhtein, T. (1993). laksesykdom for&r&et av rickettsie. Norsk Fiskeoppdmtt NR. 1240-41.

(conrinued)


Table 3. (Conrinued)

Schafer, J.W., Alvarado, V., Enriquez, R., Monds, M. (1990). The coho salmon syndrome (CSS): a new disease in Chilean salmon, reared in sea water. Bull. Eur. Assoc. Fish Pathol. 10:130.

Smith, P.A., Lannan, C.N., Games, L.H., Jarpa, M., Larenas, J., Caswell-Reno, P., Whipple, M., Fryer, J.L. (1995). Piscirickettsiosis: A bacterin field trial in coho salmon (Oncorhynchus kiturch). Bull. Eur. Assoc. Fish Pathol. 15(4):137-141.

Wada, S., Hatai, K., Shiomitsu, T. (199.5). A disease with liver swelling and nodule formation in cultured tilapia. (Abstract in Japanese) 1995 Spring Meeting Japan. Sot. Fish Pathol., March 30-31, 1995, Tokyo, Japan

Wolf, K. (1981). Chlamydia and rickettsia of fish. Fish Health News 10(3):1-5.

this disease resulted in excessive mortality, reaching 95% in some cases. Khoo et al. (14) observed rickettsia-like organisms in moribund specimens of a tropical freshwater fish, the blue-eyed plecostomus Punuque suttoni, shipped to the United States from Colombia. In France, Comps et al. (15) found rickettsia-like organisms in the brain of moribund juvenile sea-bass (Dicentrurchus l&-ax) exhibiting abnormal swimming behavior. In Chile, Gaggero et al. (16) isolated J? salmonis from diseased coho salmon and rainbow trout held only in fresh water. Also in Chile, a different and unidentified bacterium was isolated from diseased Atlantic salmon reared exclusively in fresh water. The organism, like P salmonis, was isolated in fish cell cultures but was reportedly smaller (ca. 0.2-0.8pm vs ca. OS-1.5pm in diameter), and showed no reaction when tested with FITC-labelled polyclonal antiserum prepared against e salmonis. It was considered by the authors to be rickettsia-like, however, because it was observed within the cytoplasm of kidney and spleen cells of infected fish and could not be cultured on seven unnamed bacteriological media (17).

PISCIRICKETTSIA SALMONIS

Taxonomic position

Piscirickertsiu sulmonis is the best chyacterized of the rickettsial agents observed in and/or isolated from fish. The type strain, LF-89, was isolated from a diseased coho salmon collected from a seawater netpen in southern Chile where an epizootic was in progress. It is taxonomically placed in the order Rickettsiales, the family Rickettsiaceue, and a new genus and species described by Fryer et al. (3). It has been deposited with the American Type Culture Collection as ATCC VR 1361.

Many of the rickettsial agents observed in or isolated from fish since 1989 have been serologically identified as E sulmonis (Table 1). In our laboratory, we have tested isolates from Chilean coho salmon and rainbow trout and from Atlantic salmon reared in Chile, Canada and Norway. Although all of these isolates are morphologically similar to LF-89 (Fig. 1.) and react with polyclonal antibodies against the type strain, monoclonal antibodies differentiate among them (unpublished data). The rickettsia-like organism isolated by Cvitanich et al. (17) reportedly does not react with polyclonal antibodies against E salmonis, and the remaining rickettsia-like organisms observed in and/or isolated from fish have not been characterized sufficiently to determine their relationship to LF-89. Taxonomic placement of these agents awaits further study.


Fig. 1. Caption opposite.


Piscirickettsiosis

Piscirickettsiu salmonis produces an epizootic fish disease, piscirickettsiosis. The mortality attributed to piscirickettsiosis in salmonids ranges from extremely high (up to 90%) in Chile (18) to low (up to O-.06%) in Canada (10) and Norway (11). All species of salmonids cultured in Chile are affected by this disease, but the greatest losses are registered in coho salmon cultured in salt water. Mortality in the seawater netpens begins 6-12 weeks after the transfer of fish from fresh water (2). It peaks in the fall of the year and rises again the following spring (19).

Varied clinical signs accompany p sulmonis infection, but few are specific to piscirickettsiosis. Moribund fish collect at the surface of the water along the edges of the sea cages. These individuals are lethargic, anorectic, and dark in color (10). The gills are pale and hematocrits low (25% or less). Frequently, skin hemorrhages and lesions are among the first signs observed. These lesions range from small areas of raised scales to shallow ulcers up to 2 cm in diameter (18). Internally&e kidney is swollen and the spleen enlarged. Petechial hemorrhages are found on the swim bladder and viscera. Diagnostic ring-shaped, cream-colored lesions are present on the livers of chronically infected fish, but in acute cases, mortality may be the only gross sign of disease observed.

The histopathology associated with /? sulmonis infection has been described by various authors (10, 18, 19,20). Briefly, piscirickettsiosis produces pathological changes in most internal organs of affected fish, with the most severe changes in intestine, kidney, liver, and spleen. Areas of necrosis and inflammation are found throughout, especially adjacent to blood vessels. Epithelial hyperplasia results in lamellar fusion in the gills. Rickettsiae are commonly observed within macrophages and in the cytoplasm of other infected cells.

Characteristics of the type strain LF-89 Piscirickettsiu salmonis type strain LF-89 is a non-motile, Gram-negative, obligately

intracellular bacterium. It is predominately coccoid, ca. 0.5-l .5 pm in diameter, but also occurs as rings or pairs of curved rods. It replicates within membrane-bound cytoplasmic vacuoles in cultured fish cells (Fig. 1A) and in the cells of tissues throughout infected fish. In thin section, it displays a typical Gram-negative cell wall and the protoplasmic structure of a procaryote. It is Gimenez-negative (21) but retains basic fuchsin when stained by Pinkertons method for rickettsia and chlamydia (22), and it stains dark blue with Giemsa. It does not react with a monoclonal antibody made against the group-specific chlamydial LPS antigen (2).

Piscirickettsiu safmonis is cultivable in fish cell lines where it produces a cytopathic effect, but it does not replicate on any known cell-free media. The in vitro growth characteristics of this agent were described by Fryer et al. (2). Replication is optimal at 15-18C. retarded above 20C and below 10C and does not occur above 25C. It replicates to titers of lo6 to 10 TCIDs, ml- in fish cell cultures. The titer is decreased by 99% or more by one cycle of freeze-thaw at -70C but the addition of 10% DMSO to the freezing medium provides a cryopreservative effect. It is sensitive to a broad range of antibiotics but not to penicillin.

When Garcts et al. (5) injected ten-fold dilutions of spent medium from an LF-89-infected cell culture into groups of 40 juvenile coho salmon and groups of 30 juvenile Atlantic salmon, mortality ranged from 88 to 100% over the 42 days of the experiment. All of the typical disease

Fig. I. Piscirickefrsia salmonis replicating within cytoplasmic vacuoles in cultured CHSE-214 cells. Note dividing cells (arrows). Bar = 1 &km. A. The type strain, LF-89, isolated from Chilean coho salmon Oncorhynchus kisutch. B. Isolate ATL-4-91 from Atlantic salmon Salmo salar in Canada. C. Isolate SLGO-94 from rainbow trout Oncorhynchus mykiss in Chile. D. Isolate EM-90 from Atlantic salmon in Chile. E. Isolate NOR-92 from Atlantic salmon in Norway.


signs were present in the inoculated coho salmon, but in the Atlantic salmon, mortality was the only gross sign of disease observed. However, the mortality in both species followed a clear dose-response pattern, and LF-89 was reisolated from moribund fish in each inoculated group.

In experiments designed to test the extracellular survival of LF-89 under varied environmental conditions, Lannan and Fryer (23) found that infectivity remained for at least 14 days in preparations of semipurified LF-89 suspended in high salinity sea water at 5,10, and 15C, but infectivity was rapidly reduced in preparations suspended in fresh water. Titers dropped below the level of detection (lo* TCID,, ml-) immediately after suspension in fresh water, and no infectious material could be recovered from these preparations at that time or at any time thereafter during the two-week experimental period.

Mode of transmission

The source, reservoir, and transmission of l? salmonis remain questions of pressing importance. Although piscirickettsiosis has been observed in fish over a widespread geographic area (Table l), with the exception of the extensive, recurring epizootics in southern Chile, the infections are sporadic, and at times, few fish farms are affected in each region where they occur. No common thread, beyond the marine environment itself, is readily apparent between sites in Canada, Norway, and Ireland. It seems likely that one or more naturally occurring aquatic animals, perhaps transiently present, provide the source and reservoir of the rickettsiae infecting cultured salmonids in these diverse areas, but the identity of the alternate host(s) remains unknown.

Piscirickettsiosis has been experimentally induced by injection (5, 19) but the normal mode(s) of transmission for this agent, whether horizontal, vertical, or through a vector, has not been demonstrated. With the exception of the genus Coxiella, an intermediate host or vector is required for transmission of rickettsial disease agents in the terrestrial environment (24), but an intermediate may not be required for delivery of the pathogen in the aquatic environment, and no such host or vector has been identified for I! salmonis. It must also be noted that the extended extracellular survival time of this organism in salt water (23) may be sufficiently long to permit horizontal transmission in the marine environment without the requirement for a vector. However, the almost immediate deactivation of the rickettsia in fresh water makes direct horizontal transmission unlikely under those conditions.

Limited studies designed to demonstrate horizontal transmission have had varied outcomes. Games et al. (5) detected no evidence of horizontal transmission between injected coho salmon, dying of piscirickettsiosis, and uninjected individuals held in a cage in the same tank of flowing fresh water, but Cvitanich et al. (19) reported horizontal transmission between injected and sham-injected individuals in a low number of coho salmon held in static freshwater and seawater aquaria. Environmental conditions differed between the two sets of experiments, e.g. flowing water at a mean temperature of 10.5C vs. static water at 15C making the results difficult to compare, but they suggest that under some circumstances, horizontal transmission can take place.

Although rickettsiae are found in the gonads of infected fish, vertical transmission of P. salmonis has not been demonstrated, and the low number of reported infections in fresh water would indicate that vertical transmission, if it occurs, must be a rare event. Until definitive studies are conducted, the questions of source, reservoir, and normal mode of transmission of R salmonis are open to conjecture.

Detection and identification

Inoculation and observation of susceptible cultured fish cells provides the most sensitive method of detection of P. salmonis (25). However, isolation of an agent sensitive to low levels of


many of the antibiotics routinely used in cell culture presents a considerable challenge, as all cultures must be maintained in antibiotic-free medium or medium containing only penicillin. In addition, aseptic collection of diagnostic specimens in the field is not readily accomplished, thereby enhancing the likelihood of contamination. For this reason, preliminary diagnosis of piscirickettsiosis is normally made by examination of Gram, Giemsa, methylene blue, or acridine orange-stained kidney imprints or smears, with confirmation of identity provided by serological methods, e.g. immunofluorescence (26) or immunohistochemistry (27).

Disease control

Although P salmonis is sensitive in vitro to many of the antibiotics commonly used to combat other infectious diseases of fish, e.g. tetracycline, erythromycin, oxolinic acid (3, 19), these preparations have been of limited efficacy in the treatment of piscirickettsiosis. Antibiotic levels may not reach lethal concentrations within host cells in vivo, permitting intracellular replication of the pathogen to continue despite therapy.

In the absence of effective methods for treatment of piscirickettsiosis, attention is necessarily centered on prevention of the disease. Vaccines have been successfully used in the control of other fish diseases (28), but unfortunately, no efficacious preparations have yet been developed to protect fish against this infection. With no vaccines, no effective chemotherapeutants, and little information on the source and transmission of the infectious agent, careful adherence to good fish culture procedures is the only tool left to fish culturists for control of this disease.

Future research

Many vital questions concerning ?? salmonis and the disease it causes remain unanswered. It is critical that the source of the infectious agent be identified if the spread of piscirickettsiosis is to be controlled. Definitive studies must be conducted to clarify the normal mode(s) of transmission of P salmonis if an understanding of the pathogenesis of piscirickettsiosis is to be attained. Much effort must be directed toward a search for the reservoir of infection in both the marine and freshwater environments, and the infectivity of F! salmonis for native, nonsalmonid fish should be investigated. The apparent difference in virulence of P salmonis in Chile and that observed in salmonids elsewhere is intriguing. It must be determined if intrinsic differences in the rickettsial isolates, the host fish, the environment, or some combination of factors is responsible for this phenomenon.

Beyond the expansion of basic knowledge about I? salmonis and piscirickettsiosis, practical research is needed to find chemotherapeutants and/or treatment regimes effective in the control of the disease, and development of an efficacious vaccine to protect valuable stocks of fish is the subject of considerable research effort. In addition, the difficulties associated with isolation of P salmonis in cell culture or detection of the agent in stained preparations require that improved methods of detection and diagnosis be developed. These methods must be easier than isolation but more sensitive and accurate than current staining procedures.

The rickettsial isolates from salmonid fish in the freshwater and marine environments should be compared, and the relationships between the rickettsial isolates from salmonid fish and the isolate from tilapia should be determined. It is unknown whether the rickettsia-like agent from tilapia is a new biotype of I? salmonis, or a new species of rickettsial fish pathogen, as yet undescribed. Finally, the search for other rickettsial fish pathogens in established and new hosts should continue. It is apparent that rickettsiae are responsible for fish diseases that span a broad


host and geographic range, and the growing awareness of the potential impact of the rickettsiae on fish health ensures that more of these agents will be found.

CONCLUSIONS

A previously unsuspected class of fish pathogens was introduced with the isolation and identification of E salmonis from Chilean salmonids in 1989 (2). With the subsequent recogni- tion of the rickettsial etiology of fish diseases in a variety of locations and host species, it is now apparent that the rickettsiae are an important group of fish pathogens with the potential for a significant effect on the health of both freshwater and marine species. We are just beginning to understand the scope and impact of these agents, and further study can only emphasize their role as major factors in the infectious diseases of fish.

Acknowledgemenrs - We thank the following individuals for providing P. salmonis isolates for our study and comparison: G. Traxler. Canadian Department of Fisheries and Oceans, Nanaimo, British Columbia; H. P Melby, Norwegian National Veterinary Institute, Oslo; P A. Smith, College of Veterinary Sciences, University of Chile, Santiago; E. Madrid, Marine Harvest Chile, Puerto Montt. This work was supported in part by Oregon Sea Grant with funds from the National Oceanic and Atmospheric Administration Office of Sea Grant, Department of Commerce, under grant NA36RGO45 I, project R/FSD-22.

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