DISEASES OF AQUATIC ORGANISMS Vol. 29: 137-144,1997 1 Published May 22 Dis Aquat Org I

REVIEW

Salmonid rickettsial septicemia caused by Piscirickettsia salm onis : a review

Felipe E. Almendras, I. Carmen Fuentealba*

Department of Pathology and Microbiology, Atlantic Veterinary College, University of Prince Edward Island, 550 University Avenue, Charlottetown, PEI, Canada CIA 4P3

ABSTRACT: Rickettsial diseases affecting several fish species have emerged in the last years. The most important outbreaks have been reported among farmed salmonid and non-salmonid species. The most important economical impact on the Chilean fish farming industry is probably the rickettsial pathogen Piscirickettsia salmonis, which is the microorganism responsible of the disease salmonid rickettsial septicemia (SRS). In the present article the authors review current knowledge on the natural history, lesions, transmission, diagnosis and control of SRS.

KEYWORDS: Piscirickettda salmonis. Salmon~drickettsial septicemia . Piscirickettsiosis . Review

INTRODUCTION Piscirickettsla salmonis was the first rickettsial agent to be implicated in the aetiology of a major fish disease, Rickettsial infections in finfish have been reported in affecting several species of salmonids cultured in salt several salmonid and non-salmonid fish in fresh water water (Cvitanich et al. 1991). The systemic character of and salt water since 1939 (Tables l & 2). However, the disease motivated the proposed name salmonid rickettsia were not considered of economical impor- rickettsial septicemia, SRS (Cvitanich et al. 1991). tance until a massive outbreak was reported in Chile. Today, this disease, also known as Piscirickettsiosis, In 1989, a new disease of unknown aetiology killed is probably the most important disease affecting the approximately 1.5 million market-sized (2 kg) coho salmon industry in Chile. Recently, the Chilean salmon Oncorhynchus kisutch cultured in the area of Salmon and Trout Growers Association reported that Calbuco, southern Chile (Bravo & Campos 1989, Cvita- all cultured salmonid species may be affected by the nich et al. 1990, Fryer et al. 1990). The disease was syndrome. Coho salmon are by far the most susceptible later described as affecting Atlantic salmon Salmo species, accounting for approximately 68% of the salar, rainbow trout Oncorhynchus mykiss, and chi- outbreaks, compared with 18% for rainbow trout and nook salmon Oncorhynchus tshawytscha (Cvitanich 14 % for Atlantic salmon (Cassigoli 1994). et al. 1991). In Chile, the losses were extensive and, on certain farms, mortalities of up to 90% were reported (Bravo & Campos 1989). ISOLATION AND GROWTH OF The causative organism of the Chilean outbreaks has PZSCIRICKETTSIASALMONIS been identified as Piscirickettsia salmonis, a rickettsial organism belonging to the order Rickettsiales, family Piscirickettsia salmonis is an obligate intracellular Rickettsiaceae, and the tribe Ehrlichieae (Fryer et al. Gram-negative, spherical to coccoid, often pleomorphic, 1992). non-motile, non-encapsulated organism ranging in size from 0.5 to 1.8 pm in diameter (Cvitanich et al. 'Addressee for correspondence. E-mail: [email protected] 1991). P. saln~onis is periodic acid-Schiff (PAS), acid-

0 Inter-Research 1997 Resale of full article not permitted 138 Dis Aquat Org 29: 137-144, 1997

Table 1 Rickettsial organisms reported in freshwater finfish M m~croscopic posterior trunk (BB)and bluegill Lep- diagnosis of the organism; I:isolation of the organism in cell llnes omis macrochirus fry caudal trunk cells (BF-2),have failed to develop a Host species Country bI/I Source CPE after infection with P, salmonis and incubation at 15OC for 30 d (Fryer Non-salmonid Tetrodon fahaka Egypt M Mohamed (1939) et al. 1990, Cvitanich et al. 1991).The Oreochromis mossambicus Taiwan M Chern & Chao (1994) reasons for the observed differences Oreochromis niloticus Taiwan hI!I Chern & Chao (1994) in susceptibility among cell lines have Oreochromis aureus Taiwan M Chern & Chao (1994) not been studied. Tjlapia zillii Taiwan M Chern & Chao (1994) Tilapia hornorum Taiwan M Chern & Chao (1994) Qchlasoma managuense Taiwan Md Chern & Chao (1994) Panaque suttoni Colombiab M Khoo et al. (1995) PRESENTATIONOF SRS Salmonid Oncorhynchusmykiss Germany M Ozel & Schwanz-Pfitzner (1975) outbreaks in water Chile M Bravo (1994) Chile M/I Gaggero et al. (1995) The first outbreaks appeared in 1988 Oncorhynchus kisutch Chile M/Id Cvitanich et al. (1991) in the Huito channel, Calbuco, Chile, Chile M/1 Gaggero et al. (1995) causing massive mortalities (up to Salmo salar Chile M/I" Garces et al. (1991) 90%) and affecting only coho salmon Chile M/I Cvitanich et al. (1995) cultured in the area (Bravo & Campos aSusceptible in experimental infection 1989).Due to the unusual characteris- bFish imported from Colombia, diagnosed in USA tics of this new disease, it was initially named U.A. (unknown agent), 'coho salmon syndrome', or 'Huito disease' fast, and Girnenez-negative, but stains well with hema- (Branson & Nieto 1991, Cvitanich et al. 1991). The dis- toxylin and eosin (H&E),Giemsa, and methylene blue ease causes substantial economic losses to the salmon (Branson & Nieto 1991, Cvitanich et al. 1991). aquaculture industry of southern Chile (Bravo & Cam- Piscirickettsia salmonis has been cul- tured in fish lines maintained Table 2. Rickettsial organisms reported In saltwater finfish. M: microscopic diag- in buffered minimal essential medium nosis of the organism; I:~solatlon of the organism in cell lines (MEM) supplemented with 10% fetal bovine serum (Fryer et al. 1990, Cvi- Host species Country M/I Source tanich et al. 1991). Typically, pisci------rickettsia1 growth is determined by Non-salmonid Callionenymus lyra Wales IM Dav~es(1986) the gradual appearance of cytopathic Djcentrarchus la brax France M Comps et al. (1996) effect (CPE) in cell monolayers. Char- acterist~c CPE include cell rounding Salmonid and the development of one or more Oncorhynchuskisutch Chile M Bravo & Campos (1989) M/I Fryer et al. (1990, 1992) large vacuoles within the cytoplasm M/I Cvitanlch et a1 (1991) (Cvitanich et al. 1991, Garces et al. M Branson & Nieto (1991) 1991). Four susceptible fish cell lines M/I Garces et al. (1991) M Lannan et a1 (1991) have been derived from salmonid spe- Oncorhynchusm ykiss Chile M Cvitanich et al. (1991) cies: chinook salmon embryo (CHSE- M Fryer et a1 (1992) 214); chum salmon Oncorhynchusketa Oncorhynchus tshawytscha Chlle M Cvitanich et a1 (1991) heart (CHH-l); coho salmon embryo 41 Fryer et al. (1992) (CSE-119); and rainbow trout gonad Canada M Evelyn (1992) (RTG-2). The other cell lines are from M Brocklebank et al. (1993) warm water fish species, epithelioma Oncorhynchusgorbuscha Canada M Evelyn (1992) papulosum cyprini (EPC) from Cypri- Salmo salar Chile M Cvitanich et al. (1991) M Garces et al. (1991) nus carpio, and the fat head minnow M Fryer et al. (1992) Pirnephales promelas (FHM) line of Canada M Brocklebank et al. (1992) epithelia1 cells, exclusive of the caudal M Evelyn(1992) l Brocklebank et al. (1993) fin (Fryer et al. 1990, Cvitanich et al. Norway M Olsen et al. (1993) 1991). Conversely, 2 cell lines, i.e. Ireland M Rodger & Drinan (1993) brown bullhead Ictalurus nebulosus Almendras & Fuentealba: Salmonid rickettsia] septicemia

pos 1989, Cassigoli 1994). Epizootics typically occur salmonis with the RLO affecting non-salmonid fish 10 to 12 wk after healthy fish are introduced into (Tilapia sp. and Plecostomus sp.). seawater, usually from March through August, and the outbreaks may last up to 10 wk and then subside (Cvitanich et al. 1991). The disease has also been de- PATHOLOGY scribed in several species of salmonids cultured in salt water in southern Chile, including Atlantic salmon, Clinical signs and gross external lesions rainbow trout, and chinook salmon (Cvitanich et al. 1991, Lannan & Fryer 1993). The clinical signs and gross lesions d.escribed for The geographical distribution of this condition may natural and experimentally infected fish are similar be wider than previously estimated since similar (Cvitanich et al. 1991). Reported clinical signs are pathogenic rickettsial organisms have been recently lethargy, anorexia, darkening of the skin, respiratory reported affecting salmonids cultured at saltwater sites distress, and surface swimming (Branson & Nieto 1991, in Canada (Brocklebank et al. 1992, 1993), Ireland Cvitanich et al. 1991). Skin lesions include perianal (Rodger & Drinan 1993), and Norway (Olsen et al. and periocular haemorrhages, petechia in the abdomen, 1993). The mortalities in these countries did not reach shallow haemorrhagic ulcers varying in size from the importance and prevalence of the Chilean out- 0.5 to 2 cm in diameter, and firm white nodules mea- breaks, and lt is not clear whether the agent producing suring up to l cm in diameter (Branson & Nieto 1991, these outbreaks was Piscirickettsia salmonis or not. Cvitanich et al. 1991). Additionally, bilateral exoph- However, isolates from the Canadian and Irish out- thalmia and ulcerative stomatitis have been described. breaks reacted positively with a polyclonal antibody However, clinical signs and external lesions are occa- made against P. salmonis (Brocklebank et al. 1993, sionally absent in affected fish (Branson & Nieto 1991, Alday-Sanz et al. 1994). Cvitanich et al. 1991).

Natural SRS outbreaks in fresh water Gross internal lesions and histological changes

Initially SRS was only described as occurring in salt The most characteristic lesions observed in heavily water (Bravo & Campos 1989, Fryer et al. 1992), al- infected fish are off-white to yellow subcapsular though subsequently natural freshwater outbreaks of nodules, measuring up to 2 cm in diameter, scattered SRS have been reported in Chilean rainbow trout and throughout the liver (Cubillos et al. 1990, Branson & coho salmon cultured in a freshwater lake (Bravo Nieto 1991, Cvitanich et al. 1991, Garces et al. 1991, 1994a, b, Gaggero et al. 1995). The lesions observed in Brocklebank et al. 1993). Macroscopic changes in affected fish and the in vitro growth characteristics of other organs include ascites, peritonitis, general pallor, the isolate were similar to those observed previously in diffuse swelling and presence of multifocal pale areas saltwater outbreaks (Gaggero et al. 1995). in the kidney and spleen (Bravo & Campos 1989, Isolation of an apparently new rickettsia-like organ- Cubillos et al. 1990, Branson & Nieto 1991). In coho ism (RLO) from Atlantic salmon has recently been salmon, the renal lesions have been interpreted as reported in Chile in fish held in fresh water, salt water chronic damage characterized by fibrosis (Cubillos et and an estuary (Cvitanich et al. 1995). Based on their al. 1990). Petechiae and ecchymoses on the serosal findings, the aetiologic agent of these new outbreaks surfaces of the pyloric caeca, swim bladder and caudal appears to be different from Piscirickettsia salmonis. intestine have also been reported in Atlantic salmon Apparently, this pathogen, U.A.2 (unknown agent 2), (Brocklebank et al. 1993). had important differences from the typical strain: it did Histological changes, have been usually classified not react with a rabbit anti-P. salmonis polyclonal anti- in the broad category of necrosis and inflammation body; presented either an extra and intracellular loca- (Garces et al. 1991). Commonly affected organs are tion; and grew in BB and BF-2 cell lines, both of which liver, spleen, intestine and hematopoietic tissue of the were previously reported as non-susceptible to P. kidney (Garces et al. 1991). Specific lesions in these salmonis (Fryer et al. 1990, Cvitanich et al. 1991, 1995). organs include multifocal to generalized coagulative In Scotland, an RLO has been isolated from farmed necrosis, presence of fibrin thrombi within small blood Atlantic slamon, associated with neurological signs vessels with necrosis of the endothelium, and infiltra- and mortality (Grant et al. 1996). Although the geo- tion by inflammatory cells (Branson & Nieto 1991, graphical distribution of freshwater outbreaks of RLO Cvitanich et al. 1991). is not limited to salmonids (Chern & Chao 1994, Khoo Microscopic lesions have also been described in et al. 1995), no information is available comparing P. other organs such as brain, skeletal muscle, skin, 140 DISAquat Org

heart, intestine, gills and ovary (Cvitanich et al. 1991). plasma membrane or wlth a nppled appearance (Fryer Specific h~stological lesions include mild to severe et a1 1990, Cvitanlch et a1 1991) These membrane pencarditis, mild endocarditis and focal hyaline necro- alterations may have been caused by shrinkage of the sis of the myocardium (Cvitanich et a1 1991) Necrosis cytoplasm durlng flxatlon and embedding procedures and inflammation of the lamina propia of the intestine, Many organisms contained one or more electron- epithellal hyperplasla of the gill, fusion of secondary lucent spherical structures (Fryer et a1 1990) Electron- lamella, and presence of RLO within the secondary dense areas containing nbosome-like structures dis- lamella blood spaces have also been detected (Branson persed throughout the cell, and fibrillar DNA-l~ke & Nieto 1991).The same authors describe renal tubular materlal was localized in the central reglon (Cvltanich degeneration and pyogranulomatous myositis with et a1 1991) Small electron-lucent vacuoles, vanable in ulceration of the overlying epidermis. Additionally, size and apparently lacklng any membrane were also meningoencephalitis, focal pyogranulomatous bran- observed In some cells (Cvitanlch et a1 1991) The chitis, pyogranulomatous splenitls w~thacute vasculitis mechanisms by whlch Pisanckettsia salmonis Infects and haemorrhage were detected in the Canadian out- the target cells, avoid the intracellular kllling activity, break (Brocklebank et al. 1993). and survlve lnslde the host are not clear and further The rickettslal organlsm infects a wide variety of studies are required cells, Including circulating macrophages, in which they replicate within variable-sized, membrane-bound, intracytoplasmic vacuoles (Cvitanich et al. 1991). Al- Pathogenesis of SRS though varying numbers of organisms are frequently observed within these intracytoplasmic vacuoles, Pis- The pathogenesis of SRS, or that caused by any other cirickettsia salmonis has also been found extracellu- aquatlc rickettsia1 organisms, has not been clarified larly as a result of cell lysis (Branson & Nieto 1991, CVI- yet. Histopathological findings of naturally and ex- tanich et al. 1991). perimentally infected fish have been described in Mixed infections with Piscirickettsia salmonis, along advanced stages of the disease (Branson & Nieto 1991, with Renibacterium salmoninarum, the causative agent Cvitanich et al. 1991), but no description of the of bacterial kidney disease (BKD), and a pathogenic sequence of histological changes and systemic dissem- microsporidian have been observed in seawater cul- ination of Piscirickettsia salmonis has been published. tured salmonids in Chile (Cvitanich et al. 1991, Smith et al. 1995). Recently, the microsporidlan Enterocyto- zoon salmonis has been reported as occurring in U.A.2 TRANSMISSION OF SRS infected fish (Cvltanich et al. 1995). Despite the high impact of RLOs on the Chilean salmon industry, the mode of invasion into the host and Infection and intracellular survival the horizontal transmission of RLO in the natural envi- ronment still remain unclear (Cvitanich et al. 1991, The location and distribution pattern of Piscirick- Garces et al. 1991, Bravo 199413). ettsia salmonis have been observed by electron micro- scopy in cell lines such as CHSE-214 (chinook salmon embryo) by Fryer et al. (1990) and Cvitanich et al. Route of infection (1991). Using scanning electron mlcroscopy, 24 h after inoculation, microorganisms of 1 pm in diameter were The intraper~toneal(1.p.) route has been reported as observed attached to the exterior surfaces of CHSE- being effective in produc~ngpiscirickettsiosis in exper- 214 cells, and after 8 d of incubation the organisms imentally infected coho and Atlantlc salmon (Cvitanlch were spilling from ruptured host cells or in the Inter- et al. 1991, GarcGs et al. 1991). Exammation of tissues cellular spaces (Fryer et al. 1990). Tissues from natu- from experimentally i p infected coho salmon showed rally and expenmentally infected fish had either indi- similar lesions to naturally infected fish (Cvltanich et vidual or paired coccoid, often pleomorphic organisms, al. 1991). usually enclosed within membrane-bound cytoplasmic Natural outbreaks of SRS typically occur a few vacuoles, with some cellular debris In addition to the weeks after smolts are transferred to the sea (Fryer et RLO (Cvitanich et al. 1991) Organisms undergoing al. 1990, Branson & Nieto 1991, Cvitanich et al. 1991), binary flssion were also observed (Cvltanich et al. 1991) which suggests that the oral route might be especially Two membrane layers were identified in these important in the case of Piscirickettsia .salmonis. organisms, a closely apposed inner membrane, and an Whether other routes of ~nfectionare important In the external layer (cell wall), usually separated from the transmission of this disease IS still unknown. Almendras& Fuentealba. Salmonid ncketts~dlsept~cemia 141

Horizontal transmission gerlings coming from positive broodstock were P, sal- monis positive. In contrast, only 26.7% of the finger- Natural horizontal transmission in seawater has been lings coming from negative broodstock were positive reported in stocks of salmon. Mortality occurred 2 wk (Bustos et al. 1994). Due to the risk of congenital or after their introduction into infected sites, although the true vertical transmission, the Chilean salmon farming mechanisms of spread are unknown (Bravo 1994b). industry has been screening broodstocks using avail- Elucidation of the natural mode(s) of infection and able detection techniques (Casslgoli 1994). transmission of Piscirickettsia salmonis are one re- search priority (Lannan & Fryer 1993). It is still unclear if transmission is direct, or through an intermediate Diagnostic methods for SRS host (i.e. sea lice, isopods) as occurs with other patho- genic rickettsial organisms (Cvitanich et al. 1991, Gar- Salmonid rickettsial septicemia is usually diagnosed ces et al. 1991).Studies in aquaria regarding horizontal from gross lesions, and with the use of histochemical transmission have shown different results. Garces et stains such as H&E, Gram, Giemsa, acridine orange, al. (1991) did not observe horizontal transmission in a Gimenez, Machiavello, and PAS to detect the pathogen group of non-inoculated coho salmon held in the same in smears or tissue sections (Fryer et al. 1990, Branson & tank with i.p. injected fish. However, Cvitanich et al. Nieto 1991, Cvitanich et al. 1991, Lannan & Fryer 1991). (1991) showed that horizontal transmission of the RLO Although these techniques are nonspecific, they are can occur in coho salmon held in seawater or fresh fast, and widely used for diagnosis of SRS. water without parasite vectors. The presence of the More specific techniques are also available. Pisci- pathogen was demonstrated using light microscopy rickettsia salmonis can be isolated in cell lines (Lannan and isolation of the RLO from mortalities of experimen- & Fryer 1991), but the technique is time consuming tally inoculated and non-inoculated cohabitant fish. and difficult since culture has to be performed without Abundant RLO-laden cells have been detected in antibiotics, which opens the possibility of other agents the intestine of coho salmon, leading to the hypothesis proliferating and contaminating the culture (Lannan et that the agent could be released through the fish al. 1991, Bustos et al. 1994). faeces, and survive to infect other fish (Cvitanich et al. Immunofluorescent antibody technique and immuno- 1991). However, in vitro experiments examining extra- histochemistry with peroxidase-anti peroxidase (PAP) cellular survival of Piscirickettsia salmonis from coho using a rabbit anti-Piscirickettsia salmonis polyclonal salmon indicated that no infectious particles could be antibody are alternative procedures to detect this detected immediately after exposure to fresh water. pathogen (Lannan et al. 1991, Alday-Sanz et al. 1994). Conversely, when suspended in salt water, infectious Enzyme linked immunosorbent assay (ELISA) has also particles of P. salmonis were detected after 10 to 15 d been used to detect SRS (Cassigoll 1994). Nested poly- (Lannan & Fryer 1994).The capacity of P. salmonis to merase chain reaction (PCR) methods to detect the P. persist in salt water could be an important epidemio- salmonis have been recently described (Mauel et al. logical factor for the transmission of the microorganism. 1996). These PCR procedures are fast, specific and Intermediate vectors may also play an important role highly sensitive for detecting P. salmonis genomic in the natural transn~ission of SRS, as it occurs in mam- DNAfrom cultured fish cells and tissues of asyntomatic mals where rickettsial diseases are mainly transmitted carriers (Mauel et al. 1996). by ticks (Woldehiwet & Ristic 1993). External parasites, such as the hematophagous isopod Ceratothoa gau- dichaudii, are commonly found affecting cultured CONTROL OF SRS salmonids in Chile (Inostroza et al. 1993, Sievers et al. 1995). Recently, Piscirickettsia salmonis was detected Chemotherapy by an indirect fluorescent antibody technique (IFAT) in tissue sections of C. gaudichaudii (Garces et al. 1994); In vitro tests have shown sensitivity to strepton~ycin, however, the importance of this finding in the trans- gentamicin, tetracycline (Fryer et al. 1990), chloram- mission of the disease requires further study. phenicol, erythromycin, oxytetracycline, clarithro- mycin and sarafloxacin (Cvitanich et al. 1991), and resistance to penicillin (Fryer et al. 1990), penicillin G, Vertical transmission and spectinomycin (Cvitanich et al. 1991). In practice, variable results have been achieved using oral antibi- Recently, vertical transmission of Piscirickettsia sal- otics (Cassigoli 1994). Losses due to the pathogen have monis was reported in coho salmon by Bustos et al. increased progressively and are apparently due to (1994). Results of IFAT showed that 98.3% of the fin- antibiotic resistance (Bustos et al. 1994, Cassigoli 142 Dis Aquat Org 29: 137-144, 1997

1994) Among the antibiotics currently used, the Cassigoli 1994). Fish carrying Piscirickettsia salmonis quinolones are the most popular, oxolinlc acid and are observed in sea cages with neither clinical signs flumequine being the maln quinolones currently used nor mortalities (Branson & Nieto 1991). It would appear orally to control outbreaks (Cassigoli 1994). that other factors are needed to precipitate the massive Although resistance to quinolones can not be dis- losses which have been experienced (Branson & Nieto carded, there are other factors that may influence the 1991). Outbreaks have occurred after smolt transfer, efficacy of the treatments. A recent report showed that water temperature changes and severe storms (Bran- several aquaculture antibacterials are antagonized by son & Nieto 1991). In salmonid populations with a high seawater cations (Barnes et al. 1995). The in vitro min- prevalence of P. salmonis infection, the decrease of imum inhibitory concentrations of oxolinic acid, fluoro- unnecessary stress and general husbandry practices quinolones, and oxytetracycline were increased 40- to such as grading, sampling, and net changes have 60-fold when esposed to MgZ+,and the inactivity of proven to be effective preventive measures for limit- antibiotics in the intestine of salmon held in the marlne ing outbreaks (Cassigoli 1994). environmerd may be especially important due to Early elimination of dead and clinically diseased the large amounts of seawater ingested (Barnes et fish, appropriate disposal of blood from harvested fish, al. 1995). Variations from pharmaceutical companies reduction of stocking densities, a decrease biomass per regarding effective dose and period of treatment have site and region, and provision of periods of site fallow- al.so been observed (Cassigoli 1994). ing are some management practices suggested by the Other quinolones, such as enrofloxacine a ay er@)and Salmon and Trout Growers Association of Chile (Cas- danofloxacin (Pfizero), are used as i.p. injections. En- sigoli 1994). Strategic measures such as examination, couraging results have been achieved with this proce- diagnosis of infected broodstock and removal of their dure, possibly because every fish receives a therapeu- eggs, and individual batch incubation of eggs are pro- tic dose (Cassigo1.i 1994). Oxytetracycline, spiramycine posals made by Bustos et al. (1994) based on their and florfenicol have also been used (Cassigoli 1994). observations of vertical transmission. Strategies to decrease the vertical or congenital transmission of Piscirickettsia salmonis include i.p. injection of broodstock with antibiotics 30 to 60 d RICKETTSIAL ORGANISMS IN NON-SALMONID before spawning, and incorporation of a.ntibiotics in FINFISH the water during hardening of the eggs after fertiliza- tion (Bustos et al. 1994). Salmonids have not been the only target of rick- ettsial organisms and several reports have been pub- lished describing rickettsia1 infections in non-salmonid Vaccines finfish (Tables 1 & 2). Recently, a RLO (0.86 X 0.63 pm) was identified as the causative agent of an outbreak There is a general agreement that a vaccine would with mass mortality among pond-reared tilapia in help considerably to control SRS (Cassigoli 1994), and Taiwan (Chern & Chao 1994). Affected species in- several groups have established research programs cluded Oreochromis mossambicus, 0, nilotirus, 0. aimed at developing a vaccine in Chile (J. Casslgoli aureus, Elapia zillii. T hornorum, and some hybrids. pers, comm.) and Canada. Only one vaccine trial using Fish had hepatic lesions charactenzed by diffuse a bacterin has been published (Smith et al. 1995) and necrotizing hepatitis, severe vasculitis, fibrin thrombi an immunoprotective effect was reported in vacci- and granuloma formation. The RLO were observed nated fish compared to controls. Although the results inside macrophages near or in the centre of granulo- obtained are encouraging, they must be interpreted. mas, especially in the spleen and kidney. Effacement cautiously since the natural challenge may have been. of hematopoietic and renal tissue by inflammatory low and Renibactenum salnloninarum was detected in cells was often observed. In gills, proliferation of experimental fish. The trial was also too short to deter- epithelia1 cells resulted in fusion of lamellae, and mine if a protective effect remained active in larger vacuolated macrophages containing the RLOs were fish where economic losses are more significant (Smith usually seen. Low hematocrits and increased numbers et al. 1995). of macrophages contaming RLOs were al.so reported (Chern & Chao 1.994). The RLO responsible for the tilapia outbreaks was Other methods isolated in epithelioma papulosum cyprini (EPC) carp cell line. The disease was reproduced in fish following Another important factor thought necessary for the intramuscular inoculation, and cohabitation among 8 development of SRS is stress (Branson & Nieto 1991, tilapia species and the cichlid Cichlasoma managu- Almendras & Fuentealba: Salmonid rickettsia1 septicemia 143

ense. Experimental inoculations produced higher mor- Barnes AC, Hastings T, Amyes SGB (1995) Aquaculture anti- talities at 15°C than at 30°C. No infection or disease bacterial~are antagonized by seawater cations. J Fish Dis 18:463-465 were produced in 8 other cichlid species (Chern & Branson EJ. Nieto D (1991) Description of a new disease con- Chao 1994). dition occurring in farmed coho salmon, Oncorhynchus An RLO affecting the blue-eyed plecostomus Panaque kjsutch (Walbaum), in South Amenca. J Fish Dis 14: suttoni, a pet fish imported from Colombia, has also 147-156 been described (Khoo et al. 1995). Numerous intra- Bravo S (1994a) First report of Piscinckettsja salmonis in freshwater. FHS/AFS Newsl 22:6 cytoplasmic rickettsial organisms were observed in the Bravo S (1994 b) Piscirickettsiosis in freshwater Bull Eur cells of the mononuclear phagocytic system, and in Assoc Fish Pathol 14:137 macrophages of the heart, spleen, kidney, and some- Bravo S. Campos M (1989) Coho salmon syndrome in Chile. times liver (Khoo et al. 1995). The microorganisms FHS/AFS Newsl 17:3 were within membrane-bound vacuoles, measured Brocklebank JR. Evelyn TPT, Speare DJ, Armstrong RD (1993) Rickettsia1 septicemia in farmed Atlantic and chi- approximately 0.5 pm, and possessed a trilaminar cell nook salmon In Bri.tish Columbia: clinical presentation and wall. Tufts of fibrin indicative of endothelial damage experimental transmission. Can Vet J 34:745-748 were present within the interstitium of the kidney and Brocklebank JR, Speare DJ, Armstrong RD, Evelyn TPT spleen. Granulomatous lesions, necrosis and efface- (1992) Septicemia suspected to be caused by a rickettsia- like agent in farmed Atlantic salmon. Can Vet J 33: ment of the hematopoietic elements were frequently 407-408 observed. Other parasites such as trypanosomes, nema- Bustos P, Entrala P, Montana J, Calbuyahue J (1994) Sep- todes and embryonated eggs were also observed in the ticemia rickettsial salmonidea (SRS): estudio de trans- liver, kidney, spleen and heart (Khoo et al. 1995). mision vertical en salmon coho (Oncorhynchus kisutch). In: Fundacion Chile (ed) Proceedings Primer Seminario Recently, a RLO infecting juvenile sea-bass Diocen- International: patologia y nutricion en el desarrollo de la trarchus labrax reared in floating sea cages at 12 to acuicultura. October 3-7. Puerto Montt, p 33-40 15°C in the Mediterranean coast of France has been Cassigoli J (1994) Septicemia rickettsial del salmon. In: Fun- described (Comps et al. 1996). Focal necrosis of mes- dacion Chile (ed) Proceedings Primer Seminario Interna- encephalic tissues associated with an inflammatory cional: Patologia y nutricion en el desarrollo de la acuicul- tura: factores de exito. October 3-7. Puerto Montt, p 17-20 reaction, and RLO contained within vacuoles were Chern RS, Chao CB (1994) Outbreaks of a disease caused seen in affected fish (Comps et al. 1996). by rickettsia-like organism in cultured tilapia in Taiwan. Fish Pathol 29:61-71 Comps M, Raymond JC, Plassiart GN (1996) Rickettsia-like CONCLUSIONS organism infecting juvenile sea-bass Dicentrarchus labrax. Bull Eur Assoc Fish Pathol 16:30-33 Cubillos V. Farias C, Alberdi A, Alvarado V, Schaffer JW, In the past 8 years reports of rickettsial diseases Enriquez R (1990). Caractensticas anatomopatologicas del affecting several finfish species have increased around sindrome del salmon choho, una nueva enfermedad de the world. Due to the intracellular location of this 10s salmonideos. Patol Anim 4:14-17 Cvitanich JD, Garate 0, Silva C, Andrade M, Figueroa C, organism, the control of these pathogens through the Smith CE (1995) Isolation of a new rickettsia-like organ- use of antibiotics has shown variable results. Piscirick- ism from Atlantic salmon in Chile. FHS/AFS Newsl 23: ettsia salmonis continues to cause large losses in the 1-3 Chilean salmon industry, increasing the production Cvitanich JD, Garate 0, Smith CE (1991) The isolation of a cost of the fish as a result of the increased mortalities in rickettsia-like organism causing disease and mortality in Chilean salmonids and its confirmation by Koch's postu- some stocks and the necessity of continuous use of late. J Fish Dis 14:121-145 antibiotics. Cvitanich JD. Garate 0, Smith CE (1990) Et~ological agent No comparison between isolates from different in a Ch~leancoho salmon disease isolated and confirmed countries have been reported. Further information by Koch's postulates. FHS/AFS Newsl 18:l-2 Davies AJ (1986) A rickettsia-like organism from dragonets, regarding horizontal and vertical transmission, patho- Callionymus lyra L. (Teleostei Callionimidae), in Wales. genesis, intracellular survival, and immunogenesis is Bull Eur Assoc Fish Pathol 6:103-104 needed in order to establish control strategies for SRS. Evelyn TPT (1992) Salmonid Rickettsial Septicemia. In: Kent Identification of risk factors in the production cycle ML (ed) Diseases of seawater netpen-reared salmonid may help to avoid unnecessary management that may fishes in the Pacific northwest. Can Spec Pub Fish Aquat Sci 116:18-19 induce outbreaks in prevalent populations. Fryer JL, Lannan CN, Garces LH, Larenas JJ, Smith PA (1990) Isolation of a rickettsiales-like organism from diseased coho salmon (Oncorhynchus kisutch) in Chile. Fish Pathol LITERATURE CITED 25:107-114 Fryer JL, Lannan CN, Giovannoni SJ, Wood ND (1992) Piscl- Alday-Sanz V, Rodger H, Turnbull T, Adams A, Richards, RH rickettsia salmonis gen. nov., sp. nov., the causative agent (1994) An immunohistochemical diagnostic test for rick- of an epizootic disease in salmonid fishes. 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Responsible Subject Editor: D. W. Bruno, Aberdeen, Manuscript first received: July 7, 1996 Scotland, UK Revised version accepted: January 14, 1997