DISEASES OF AQUATIC ORGANISMS Vol. 32:49-69,1998 1 Published February 26 I Dis Aquat Org

REVIEW Occurrence and significance of atypical Aeromonas salmonicida in non-salmonid and salmonid fish species: a review

Tom WiklundL',Inger ~alsgaard~

'Institute of Parasitology and Department of Biology, Abo Akademi University, BioCity. Artillerigatan 6. SF-20520 Abo. Finland 'Danish Institute for Fisheries Research. Fish Disease Laboratory. Biilowsvej 13, DK-1870 Frederiksberg C. Denmark

ABSTRACT. Bacterial strains of Aeromonas salmonicida included in the recognized subsp. achromo- genes, subsp. masoucida, and subsp. smithia in addition to the large number of strains not included in any of the described subspecies are referred to as atypical A. salmonicida. The atypical strains form a very heterogeneous group w~threspect to biochemical characteristics, growth conditions, and produc- tion of extracellular proteases. Consequently, the present taxonomy of the species A. salmonicida is rather ambiguous. Atypical A. salmonicida has been isolated from a wide range of cultivated and wild fish species, non-salmonids as well as salrnonids, inhabiting fresh water, brackish water and marine environments in northern and central Europe, South Africa. North America. Japan and Australia. In non-salmonid fish species, infections with atypical strains often manifest themselves as superficial skin ulcerations. The best known diseases associated with atypical A. salmonicida are carp Cypnnus carpio erythrodermatitis, goldfish Carassius auratus ulcer disease, and ulcer disease of flounder Platichthys flesus, but atypical strains are apparently involved in more disease outbreaks than previously sus- pected. Macroscopical and microscopical studies of ulcerated fish indicate internal organs are infre- quently invaded by atypical A. salmonicida. This view is supported by the fact that atypical strains are ~rregularly isolated from visceral organs of ulcerated fish. High mortality caused by atypical A. salmonicida has been observed in populations of wild non-salmonids and farmed salmon~ds,although the association between the mortality in the wild fish stocks and atypical A. salmonicida has not always been properly assessed. In injection experiments the pathogenicity of the atypical strains examined showed large variation. An extracellular A-layer has been detected in different atypical strains, but virulence mechanisms different from those described for (typical) A. salmonicida subsp. salrnonicida, for example an extracellular metallo-protease and a different iron utilization mechanism, have been described. Limited information is available about the ecology, spread and surv~valof atypical strains in water. The commonly used therapeutic methods for the control of diseases in farmed fish caused by atypical A. salrnonicida are generally effective against the atypical strains. Resistance to different antibiotics and transferable plasmids encoding multiple drug resistance have been observed in atypi- cal A. salmonicida. Studies aimed at producing a vaccine against atypical strains are in progress.

KEY WORDS: Aeromonas salmonicida . Slun ulcers . Fish pathogens . Fish

INTRODUCTION cies A. salmonicida but showing biochemical character- istics, mainly slow growth and late pigment production, The expression 'atypical Aeromonas salmonicida' was different from those described for A. salmonicida subsp. initially used for bacterial strains belonging to the spe- salrnonicida. One of the first papers using the expression 'atypical' or 'aberrant' strain of A. salrnonicida was pub- lished in 1971 (Evelyn 197 although reports on strains 'Present address: Danish Institute for Fisheries Research, Fish l), Disease Laboratory, Biilowsvej 13, DK-1870 Frederiksberg C. not included in subsp. salmonicida had been published Denmark. E-mail: [email protected] earlier (Smith 1963, Kimura 1969a, b).

O Inter-Research 1998 Resale of full article not perrmtted 50 Dis Aquat Org

Although several subspecies of Aeromonas salmoni- This paper presents the current state of knowledge cida have been described, i.e. subsp. achromogenes. about the biology and the taxonomy of atypical subsp. n~asoucida, subsp. salrnonicida and subsp. Aeromonas salmonicida strains and disease processes smlthia (Holt et al. 1994), a large number of reported generated by these bacteria. strains have not been ass~gnedto any of these sub- species. Presently, strains included in the subsp. achro- mogenes, subsp. rnasouc~da, and subsp. smithia in TAXONOMY addition to the strains not included in any of the described subspecies of A. salmonicida are referred to The taxonomic position of Aeromonas salmonicida as atypical strains. Strains of subsp. salrnonicida are has been reviewed by McCarthy & Roberts (1980) and consequently referred to as 'typical' A. salmonicida. Austin & Austin (1993). In Bergey's Manual of System- Although strains showing characteristics that are dif- atic Bacteriology (Popoff 1984), the genus Aeromonas ferent from those described for typical A. salmonicida was included in the family Vibnonaceae. The proposal are considered to be atypical isolates, several papers to place this genus in the new family Aeromonadaceae on typical strains have recently been published show- (Colwell et al. 1986), which was based on molecular ing atypical or aberrdnt biochemical characteristics, genetic data, has recently been validated (Anonymous such as non- or late-pigmentation (Wiklund et al. 1992). The recognized species in the genus Aeromonas 1993), negative cytochrome oxidase reaction (Chap- represent 14 clearly different DNA homology groups man et al. 1991), acid product~on from sucrose (Wik- (Esteve et al. 1995) with A. salmonicida subsp. sal- lund et al. 1992), no degradation of aesculin (Austin et monicida belonging to DNA group 3. al. 1989), production of hydrogen sulfide (Austin et al. According to the latest edition of Bergey's Manual 1989) and growth at 37°C (McIntosh & Austin 1991). of Systematic Bacteriology (Popoff 1984), Aeromonas The number of published reports of disease out- salmonicida is divided into 3 subspecies: A. salmoni- breaks associated with atypical strains has increased cida subsp. saLmonicida (Griffin et al. 1953a), A. significantly during the last decade, and these isolates salmonicida subsp. achromogenes (Smith 1963) and A. have been reported from an increasing number of fish salmonicida subsp. rnasouclda (Kimura 1969a, b). This species and geographical areas. Most of the isolated differentiation was proposed by Schubert (1967, 1969). strains have been identified as atypical Aerornonas These 3 subspecies can be distinguished by a limited salmonicida and very few of them are included in any number of biochemical characteristics (Popoff 1984). of the described subspecies (subsp. achromogenes, Since the first descript~onby Smith (1963) of strains subsp. masoucida, and subsp. smithia) (Holt et al. different from Aeromonas salmonicida subsp. sal- 1994). In fact, new isolates of A. salmonicida subsp. monicida, a large number of atypical strains of A. masoucida and subsp. smithia have not been reported salmonicida have been isolated (e.g. Wiklund 1990, since their initial isolation and description (Kimura Austin & Austin 1993, Nakatsugawa 1994, Wiklund & 1969a, b, Austin et al. 1989). Dalsgaard 1995). McCarthy & Roberts (1980) have Diseases caused by atypical strains have been proposed another and different division into 3 sub- referred to as ulcer disease or 'atypical furunculosis' species, ba.sed on epizootiological criteria. The 3 sub- (Snieszko et al. 1950, Hubbert & Williams 1980, Gro- species are subsp, salrnon~cida, subsp, achromogenes man et al. 1992, Wiklund 1994b, Gudmundsdottir & (incorporating subsp, masoucida, including only strains Magnadottir 1997) in contrast to furunculosis or classi- isolated from salmonids), and subsp, nova (isolated cal furunculosis caused by typical Aeromonas salmoni- from non-salmonid fish). This classification has not cida. Unfortunately, clinical infections in fish with been approved in Bergey's Manual of Systematic Bac- atypical A. salmonicida have sometimes in the litera- teriology (Popoff 1984). A. salmonicida subsp. salmoni- ture been referred to as furunculosis (McCarthy 1975, cida and A. salmonicida subsp. masoucida were found Bucke 1980, Boomker et al. 1984), which in the oplnion to be a genetically homogenous group in DNA homo- of the present authors should be avoided. logy studies (MacInnes et al. 1979). Based on numeri- Atypical bacterial strains of the species Aeromonas cal taxonomy (McCarthy 1978), sequence homology, salmonicida have been poorly defin.ed, and this group and guanine-plus-cytosine content (%G+C) (Belland consists of isolates showing a large variety of bio- & Trust 1988), the subspecies achromogenes should chemical, molecular and virulence characteristics. be restructured to include the present subspecies Presently, an atypical strain can be defined only as a masoucida. The close genetic relatedness of these sub- strain that does not fit into the existing classification of species was also shown by Martinez-Murcia et al. A. salmonicida subsp. salmonicida. This situation cer- (1992), in contrast to the findings of Austin et al. (1989), tainly needs to be adequately solved in future detailed which supported the separation of the 2 subspecies. taxonomic studies. However, in Bergey's Manual of Determinative Bac- Wiklund & Dalsgaard: Atypical Aeromonas salmonicida: a review 51

teriology (Holt et al. 1994) the 3 subspecies from the biocin (5 pg) (Magarinos et al. 1992). With respect to approved list (Skerman et al. 1980) are included motility, the genus consists of 2 well-separated groups together with a new subgroup, subsp. smithia, which with the species and subspecies of Aeromonas sal- was created by Austin et al. (1989). monlc~dabelonging to the non-motile group. The opti- In the description of Aeromonadaceae (Colwell et mal growth temperature for A. saln?onicida is 20 to al. 1986) the %G+C content of the DNA ranged from 22°C (Paterson et al. 1980a). Growth may be accompa- 40 to 63 mol%. The %G+C contents of the DNA of sub- nied by the release of a brown pigment which is a species of Aeromonas salmonicida in different studies property used in the classification of A. salmonicida ranged from 52 to 67 moll%, (Table 1). The %G+C subspecies (Griffin et al. 1953b, Donlon et al. 1983, Alt- values given in Table 1 do not allow for distinction of mann et al. 1992). The existence of achromogenic or the different subspecies. slowly pigmenting strains of A. salmonicida has been Although several studies have described the genetic described, with the consequence that different strains relationships between the species within the genus have not been properly identified (Bulkley 1969, Maw- Aeromonas, there is still a lack of reliable traits for sub- desley-Thomas 1969, LeTendre et al. 1972, Michel species discrimination, especially for the slow-growing 1981). fastidious strains which have been isolated during According to Schubert (1967), Aeromonas salmoni- the past few years (Pedersen et al. 1994, Wiklund et cida subsp. achromogenes differs only in a few proper- al. 1994). Further studies have to be based on large ties from subsp. salmonicida. Identification of atypical numbers of strains and use more modern techniques A. salmonicida strains to the genus level is easily such as poly-nucleotide sequencing and DNA-DNA or achieved with sufficient experience. However, reports RNA-DNA hybridization. published on the isolation of cytochrome oxidase neg- ative atypical as well as typical strains (Traxler & Bell 1988. Chapman et al. 1991, Olivier 1992, Pedersen et IDENTIFICATION AND CHARACTERIZATION al. 1994, Wiklund et al. 1994) have complicated identi- fication. In addition, the fermentative reaction of glu- The genus Aeromonas (Kluyver & Van Niel 1936) cose in Hugh & Leifson's medium sometimes requires consists of a collection of oxidase- and catalase-positive, prolonged incubation, especially for the identification glucose-fermenting, facultatively anaerobic, Gram- of the slow-growing fastidious strains isolated from negative, rod-shaped bacteria (Popoff 1984) that are flatfish (Pedersen et al. 1994, Wiklund et al. 1994). resistant to vibriostatic agent 0/129 (10 1-19)and novo- Identification to the species level can be further con- firmed by demonstrating the antigenic similarity be- tween the typical and atypical strains with respect to Table 1. Aeromonas salmonicida subspecies. Base composition the thermostable 0-antigen, by macroscopic slide of the DNA. G+C: guanine-plus-cytosine; nd: no data available agglutination using antiserum prepared from an iso- late of Aeroinonas salmonicida subsp. salmonicida Subspecies G+C contents Soul ce or group mol% (McCarthy 1977, Trust et al. 1980c, Pedersen et al. 1994) or a hybridoma producing a highly specific A. salmonicida 55.1-57.5 McCarthy (1978) monoclonal antibody against A. salmonicida (Yoshi- 62.7 MacInnes et al. (1979) 57-59 Popoff (1984) mizu et al. 1993). 52.6-37.8 Belland & Trust (1988) Biochemical characterist.ics useful for distinguishing A. achrornogenes 52.9 McCarthy (1975) the different atypical subspecies of Aeromonas sal- 59.7 Belland & Trust (1988) monicida from the homogenous and well-described A. A. masoucida 60.0 Belland & Trust (1988) salmonicida subsp. salmonicida are summarized in A. nova 56.1.-57.6 McCarthy (1978) Table 2. The identification of atypical strains and the A. srnithia 55.9 0.5 Austin et al. (1989) separation of these from subsp, salmonicida has been Phenon 13 nd Austin et al. (1989) complicated because strains of subsp, salmonicida Slow-growing 54.0-66.9 Wiklund et al. (1994) have been found to differ in the key characteristics: fastidious (flatfish) pigment production (Wiklund et al. 1993), cytochrome Atypical strains Bootsma et al. (1977) oxidase activity (Chapman et al. 1991), and acid from McCarthy (1978) Paterson et al. (1980b) sucrose (Wiklund et al. 1992). An accurate biochemical Shotts et al. (1980) identification of atypical A. salmonicida based on a Trust et al. (1980b) comparison of inter-laboratory evaluations has proven Kitao et al. (1984) Ohtsuka et al. (1984) difficult; likewise data presented in the literature on Belland & Trust (1988) various strains of A. salnlonicida are not readily com- parable (Dalsgaard et al. 1998). 52 Dis Aquat Org 32: 49-69, 1998

Table 2. Aeromonas salmonicida. Characteristlcs for differentiation between subspecies and atypical ~solates.+: positive reaction; -: negative reaction; ( ): reaction of a few strains; S: sensitive; R:resistant; nd- no data available

Subspecies Pigment Oxidase Gas from Acid from Indole Degradation Sensitivity Sensitivity to or group production reaction glucose sucrose production of aesculin to ampicillin cephalothin

A. salmonicida +(-l A. achromogenesd -(+l A. masouclda" - A. nova" -/+ A. smithia C -(+l Phenon 13' +(-l Slow-growingd - Atypical isolatese +/- "Popoff (1984). Chapman et al. (1991). Wiklund et al. (1992, 1993) bMcCarthy (1978),Bohm et al. (1986).Olivier (1992) CAustinet al. (1989) dPedersen et al. (1994),Wiklund et al. (1994),Wiklund & Dalsgaard (1995) 'Dalsgaard & Fdulsen (1986),Hirvela-Koski et al. (1994), Nakatsugatva (1994)

McCarthy (1978),Bohm et al. (1986) and Olivier (1992) HOST RANGE classified the examined atypical strains as subspecies achromogenes and nova. Some important characteristics Atypical Aeromonas salmonicida strains have been of these isolates were the same for all strains (Table 2), isolated from a wide range of fish species belonging to but subspecies nova seemed to be more fastidious and different families and orders (Table 3). More than 20 growth could require up to 5 d incubation at 22'C. farmed and 30 wild fish species have been reported to Austin et al. (1989) used numerical taxonomy and harbor atypical A. salmonicida strains. Initially, atypi- DNA:DNA hybridization techniques to group Aero- cal A. salmonicida strains were predominantly re- monas salmonicida into 5 phena, which were equated covered from fresh water or anadromous fish species. with the subspecies salmonicida, masoucida, achromo- However, more recently atypical strains have repeat- genes and smithia and a fifth group (phenon 13) com- edly been isolated from marine fish species, like flat- prising unrecognized subspecies. Further strains which fish (order Pleuronectiformes) and codfish (order Gad- were able to grow at 37°C were included in the fifth iformes), as well as catadromous fish species, like eels group (Austin 1993). (family Anguillidae) (Table 3). The plasmid profiles reported from atypical strains of Aeromonas salmonicida were different from the plas- mids carried by typical strains (Bast et al. 1988, Belland & Trust 1989, Pedersen et al. 1996), and Belland & Trust (1989) suggested that plasmid content may be a useful With regard to non-salmonid wild fish, atypical epizootiological marker for atypical A. salmonicida. Aeromonas salmonicida has been isolated from occa- The results presented by Pedersen et al. (1996) indi- sional cases of ulcerated or otherwise diseased fish cated that plasmid profiles of atypical strains may from nature or from wild fish kept in aquaria or tanks change considerably in vivo and several epizootiologi- (Evelyn 197 1, Cornick et al. 1984, Dalsgaard & Paulsen cally related strains showed different profiles. Similarly, 1986, Wiklund 1990, Wilson & Holliman 1994). In a few the variability observed in plasmids in different strains cases, atypical A. salmonicida has been associated isolated from flounder Platichthys flesus seemed to be with epizootics in wild fish populations (Bulkley 1969, of no value for epizootiological work because variability McCarthy 1975, Hastein et al. 1978, Michel 1981). occurred within the isolates from the same geographi- In several studies atypical Aeromonas salmonicida cal area (Dalsgaard 1994a). In contrast to plasmid pro- has been isolated from fish which after capture were files, ribotypes never showed variation upon repeated transferred to tanks, aquaria or net pens. This was the testing (Pedersen et al. 1996). Most epizootiologically case, for example, for sablefish Anoplopoma fimbria unrelated strains had different ribotypes, whereas iso- (Evelyn 1971), Atlantic cod Gadus morhua (Cornick et lates from the same outbreak were identical. The data al. 1984) and Pacific herring Clupea harengus pallasi obtained so far suggest that nbotyping can permit the in Canada (Traxler & Bell 1988), sand-eels Hyperoplus identification of subgroups, which may be useful in epi- lanceolatus and Ammodytes lanced in Denmark (Dals- zootiological studies (McCormick et al. 1990, Dalsgaard gaard & Paulsen 1986), and greenback flounder Rhom- 1994a, Whittington et al. 1995, Pedersen at al. 1996). bosolea tapirina in Tasmania (Whittington et al. 1995). Wiklund & Dalsgaard: Atypical Aerornonas salmonicida:a review

Table 3. Aerornonas salmonicida.lsolates of atypical A. salmonicida from farmed and wild fish species in different countries

Fish species Scientific name Country Source Order Anguilliformes FARMED FISH American eel Anguilla rostrata USA Noga & Berkhoff (1990) European eel Anguilla dnguilla Denmark'' Dalsgaard (1994b) Japanese eel Anguilla japonica Japan Ohtsuka et al. (1984) WlLD FlSH American eel Anguilla rostrata Canada. USA Olivier (1992). Noga & Berkhoff (1990) European eel Anguilla anguilla Denmark Dalsgaard (1994b) Order Clupeiformes WlLD FlSH Pacific herring Clupea harenguspallasi Canada * Traxler & Bell (1988) Order Cypriniformes FARMED FISH Carp Cyprinuscarpio Australia Humphrey & Ashburner (1993) Denmark Belland & Trust (1988) Germany Bohm et al. (1986), Mirle et al. (1986) Hungary Csaba et al. (1980a) Netherlands lshiguro et al. (1986) UK Austin (1993) Yugoslavia Bootsma et al. (1977) Goldfish Carassiusauratus Australia Trust et al. (1980c), Humphrey & Ashburner (1993) Denmark Dalsgaard (unpubl. data) Germany Bohm et al. (1986) ltaly Whittington et al. (1987) Japan Elliott & Shotts (1980a) Netherlands Evenberg et al. (1982) Singapore Whittington et al. (1995) UK Elliott & Shotts (1980a) USA Elliott & Shotts (1980a) Yusoslavia Evenberg et al. (1982) 'Shu bunkln' Carassiussp. Germany Bohrn et al. (1986) WILD FISH Bream Abramis brarna Finland Wiklund (unpubl. data) Chub Leuciscus cephalus UK Wilson & Holliman (1994) Crucian carp Carassiuscarassius Hungary Csaba et al. (1980a) Dace Leuciscus leuciscus Finland Hirvela-Koski et al. (1994) Goldfish Carassiusauratus Australia Whittington et al. (1987) Canada Munluttrick & Leatherland (1984) Minnow Phoxinus phoxin us Norway Hdstein et al. (1978) Roach Rutilus rutilus Australia Humphrey & Ashburner (1993) Finland Hirvela-Koski et al. (1994) Sweden Wichardt et al. (1989) UK McCarthy (1975) Rudd Scardinius erythrophthalmus UK Gudrnundsdottir et al. (1990) Silver bream Blicca bjoerkna UK McCarthy (1975) Order Gadiformes WILD FISH Cod Gadusrnorhua Canada" Cornick et al. (1984) Icelandd Gudmundsdottir et al. (1995) Four bearded rockling Enchelyopus clni brius Denmark Dalsgaard (1994b) Haddock Melanogramrnusaeglefinus Canadaa OLivier (1992) lceland Gudmundsdottir (1996) Tomcod Gadusmicrogadus Canada Olivier (1992) Viviparous blenny Zoarces viviparus Baltic Sea Wiklund (unpubl. data) Denmark Dalsgaard (1994b) Whiting ~Merlangjusmedangus Iceland Guamundsdottlr et al. (1996) Order Perciformes FARMED FISH Common wolf fish Anarhjchas lupus Norway a Hellberg et al. (1996) Silver perch Bidyanus bidyanus Australia Humphrey & Ashburner (1993) Spotted wolffish Anarhichas minor Norway M. Valheim (pers. comm.) Wrasse Labrus berggylta Norway A. B. Olsen (pers. comm.) WILD FISH Goldsinny wrasse Ctenolabrusrupestris Ireland Frerichs et al. (1992) Norway a Gravningen et al. (1996) Perch Perca flu via tilis ~rance~ Michel (1981) Sweden Wichardt et al. (1989) UK Bucke et al. (1979) Ammodytes lanced Denmarka Dalsgaard & Paulsen (1986) Hyperopluslanceolatus Smallmouth bassC Micropterusdolomieui USA LeTendre et al. (1972) Yellow bassC Morone mississippiensis USA Bulkley (1969)

(Table continued on next page) Dis Aquat Org 32: 49-69, 1998

Table 3 (continued)

Fish species Scientific name Country Source I Order Pleuronectiformes FARMED FISH Shotted halibut Eopsetta grigorje wi Japan Nakatsugawa (1994) Turbot Scophthalmus maximus Denmark Pedersen et al. (1994) Norway Gravningen & Dydland (1991) WILD FISH American plaice Hippoglossoides platessoides Canadad Olivier (1992) Dab Lirnanda limanda Baltic Sea Wiklund (unpubl. data) Denmark Wiklund & Dalsgaard (1995) Flounder Platichthys Iles~rs Denmark Wiklund & Dalsgaard (1995) Finland Wiklund & Bylund (1991) Southern Baltic Sea Wiklund & Dalsgaard (1995) Greenback flounder Rhombosolea tapirina Australia a Whittington et al. (1995) Halibut Hippoglossus hippoglossus Canada" Cornick et al. (1984) Norway Gudmundsdottir (1996) Plaice Pleuronert.?~pla tessa Denmark Wiklund & Dalsgaard (1995) Turbot Scoph thalmus maximus Baltic Sea Wiklund (unpubl. data) Order Salmoniformes FARMED FISH Arctic char Salvelin us alpin us Canada Olivier (1992) Finland Hirvela-Kosk~et al. (1994) Iceland Gudmundsdottir (1996) Norway M. Valheim (pers. comm.) Sweden Ljungberg & Johansson (1977) Atlantic salmon Salmo sala~ Canada Paterson et al. (1980b) Faeroe Islands Dalsgaard & Mortensen (1988) Finland Rlntamakl & Valtonen 11991) ~celand Gudmundsdottir et al. (1990j Norway Olsen et a1 (1989) Sweden Llunqberq & Johansson (1977) Brook trout Salvelin us fontinalis Canada B~LI~C~eial. (1971) Sweden Ljungberg & Johansson (1977) USA Snieszko et al. (1950) Brown trout Salmo trutta m. farin Finland Hirveld-Koski et al. (1994) ~celand Gudmundsdottir (1996) Sweden Ljungberg & Johansson (1977) Chum salmon Oncorhynchus keta Canada Evelyn (1971) Grayling Thymallus thymallus Finland Rintamaki & Valtonen (1991) Sweden Ljungberg & Johansson (1977) Lake trout Salmo trutta m. lacustris Finland Ojala (1966) Sweden Ljungberg & Johansson (1977) Masu salmon Oncorhynchus masou Japan Kirnura (1969a) Pink salmon Oncorh ynchus gorbuscha Japan Kimura (1969a) Rainbow trout Oncorhynchus m ykiss Canada Olivier (1992) Finland Hirvela-Koski et al. (1994) South Africa Boomker et al. (1984) Sweden Ljungberg & Johansson (1977) Sea trout Salmo trutta m. trutta Faeroe islands Gudmundsdottir (1990) Finland hntamaki & Valtonen (1991) Norwayb Pedersen et al. (1996) Sweden Wichardt (1983a) Whitefish Coregon us sp. Finland Hirvela-Koski et al. (1994) WILD FlSH Atl.antic salmon Saho salar Canada Olivier (1992) Grayling Thymallus thymalluc F~nland Hirvela-Koslu et al. (1994) Pike Esox lucius Finland Wiklund (1990) Sweden Wichardt et al. (1989) UK Austin et al. (1989) Sea trout Salmo trulta m. trutta Scotland Smith (1963) Sockeye salmon Oncorhynchus nerka Canadad Evelyn (1971) Order Scorpaeniformes WILD FlSH Sablefish Anoplopoma fimbria Canada" Evelyn (1971) dWild fish kept in tanks, aquaria or net pens "ot reported whether isolated from wild or farmed f~sh 'Present identification of isolates as atypical Aeromonas salmoniada is based on slow pigment production *Isolated strains subsequently identified as likely atypical Aeromonas salmonicida (C.Michel pers. comm.) Wiklund & Dalsgaard:Atypical Aeromonas salrnonjcida: a review

In most studies the fish showed signs of disease shortly Atypical Aeromonas salmonicida strains have been after transfer to the tanks or aquaria. However, the isolated from fish in fresh water (Bulkley 1969, Hdstein source of the infection usually remained unknown, and et al. 1978, Michel 1981, Whittington et al. 1987, Noga it was not definitely established whether the fish & Berkhoff 1990, Wilson & Holliman 1994), brackish infected with atypical A. salmonicida harbored the water (Wiklund 1990, Wiklund & Bylund 1991, Wiklund pathogen at the time of capture or whether the fish et al. 1994), as well as salt water habitats (Boomker et were infected during transport or from the water or al. 1984, Cornick et al. 1984, Dalsgaard & Paulsen equipment in the tanks or aquaria (Evelyn 1971, Cor- 1986, Pedersen et al. 1994, Whittington et al. 1995, nick et al. 1984, Dalsgaard & Paulsen 1986, Whitting- Wiklund & Dalsgaard 1995, Hellberg et al. 1996). ton et al. 1995). In other studies atypical strains have been isolated from the skin (Hubbert & Williams 1980), gills (Benediktsdottir & Helgason 1990), and kidney PATHOLOGY (Frerichs et al. 1992) of clinically healthy fish without any signs of disease, indicating that atypical A. sal- nlonicida might be present in or on the fish and that stressful conditions (e.g.capture and transfer to tanks) Atypical Aeron~onas salmonicida infection in non- may cause subsequent outbreaks of disease. salmonids often manifests itself predominantly as a skin infection (skin ulceration) without subsequent general septicaemia. Skin ulcerations and skin lesions on wild Salmonids and farmed non-salmonid fish have been observed in connection with most disease outbreaks associated Fish belonging to different species of the family with atypical A. salmonicida. The best described dis- Salmonidae have frequently been reported to be eases showing these symptoms are carp erythroder- infected with atypical Aeromonas salmonicida (see matitis (CE; Fijan 1972, Bootsma et al. 1977, Csaba et Table 3); increased reporting is probably due to the al. 1980a, b, Sovenyi et al. 1984. Bohm et al. 1986, Mirle intensive farming of, and economical interest in, these et al. 1986), goldfish ulcer disease (CUD; Mawdesley- species. More than 10 different salmonid species have Thomas 1969, Elliott & Shotts 1980a, Shotts et al. 1980, been found to be infected with atypical A. salmonicida. Trust et al. 1980c, Bohm et al. 1986, Whittington et al. There are, however, comparably few reports on the 1987, Humphrey & Ashburner 1993, Whittington et al. isolation of atypical strains from wild fish belonging to 1995), and ulcer disease of flounder (UDF) in the Baltic family Salmonidae (Evelyn 1971, Olivier 1992, Hirvela- and the North Sea, including adjacent rivers (Nounou Koski et al. 1994) (Table 3). et al. 1981, Moller 1990, Wiklund & Bylund 1991, U11- rich 1992, Vethaak 1992, Wiklund & Bylund 1993, Wik- lund 1994a, b, Wiklund et al. 1994, Wiklund & Dals- GEOGRAPHICAL DISTRIBUTION gaard 1995). In addition, many other fish species have been reported to be affected by ulcerations associated The geographical distribution of reported isolations with atypical A. salrnonicida, and the pathogen is ap- of atypical Aeromonas salmonicida is indicated in parently involved in more disease outbreaks than pre- Table 3. Available reports suggest that atypical A. viously suspected (McCarthy 1975, Hdstein et al. 1978, salmonicida strains infect fish mainly in the temperate Ohtsuka et al. 1984, Dalsgaard & Paulsen 1986, Noga regions of the northern hemisphere, that is, Canada, & Berkhoff 1990, Wiklund 1990, Pedersen et al. 1994, the USA, Japan, and central and northern Europe Wilson & Holliman 1994, Larsen & Pedersen 1996). including the Nordic countries. In addition, atypical In carp affected by CE, the ulcers can be found all strains have been isolated from fish in southern Aus- over the body surface except on the head (Jeney & tralia including Tasmania (Whittington et al. 1987, Jeney 1995). The scales of the fish have been reported Whittington et al. 1995), South Africa (Boomker et al. to be initially surrounded by inflammation with in- 1984) and from fish exported from Singapore (Whit- filtration of inflammatory cells. The scales are sub- tington et al. 1995). There are also a few reports of sequently shed. The epithelium and corium become atypical strains isolated from fish in the Mediterranean necrotized with marked hyperaemia surrounding the area: from carp Cyprinus carpio and goldfish Carassius resulting ulcer. The inflammatory cells subsequently auratus in former Yugoslavia (Bootsma et al. 1977, spread into the muscle (Gayer et al. 1980, Jeney & Evenberg et al. 1982) and from goldfish in Italy (Whit- Jeney 1995). tington et al. 1987). To date, there is no information Mawdesley-Thomas (1969) reported that the general available about atypical A. salmonicida infecting fish health of goldfish infected with Aeromonas salmonicida in South America, Russia or northern Asia. was variable. Affected fish developed skin ulcers of 56 Dis Aquat Org32: 49-69, 1998

varying size and depth, which could appear on any part ulcers (Noga & Berkhoff 1990). Many lesions had ex- of the body. In some specimens even the face, opercu- tensive collagen deposition, contributing to the tissue lum and the eyes were affected. A marked increase in swelling. Bacterial microcolonics wcrc common on the pigmentation in and around the lesions and inflamma- surface and within ulcerations (Noga & Berkhoff 1990). tory cells penetrating far into the surrounding muscula- In addition to skin ulcerations, atypical Aeromonas ture were observed. Some of the ulcers were secondar- salmonicida has also been associated with erosions on ily infected with Saprolegnia sp. Some fish died within the mouth, varying from weak haemorrhage on the a few days; others showed lethargy, loss of orientation snout (sand-eels; Dalsgaard & Paulsen 1986), and ero- and abnormal swimming movements. sion of the lip (shotted halibut Eopsetta gngo jewi; In flounder affected by UDF, the ulcers have been Nakatsugawa 1994) to destruction of the bones in the described as open, dark red, rounded, and superficial upper and lower jaws (minnow Phoxinus phoxinus; (Wiklund & Bylund 1993). Ulcer size varied from small Hdstein et al. 1978) of non-salmonid fish. Additionally, lesions (1 to 2 mm) to large eroded areas (10 to 20 mm haemorrhage and erosion of the fins (McCarthy 1975, in diameter). The development of the ulcers was Trust et al. 1980c. Wilson & Holliman 1994, Jeney & divided into 3 stages: (1) initially weak haemorrhage in Jeney 1995),with subsequent necrosis of the tail (Dals- the skin which developed into (2) a white lesion sur- gaard & Paulsen 1986), and haemorrhage or lesions in rounded by a red rim of haemorrhagic inflammatory the eyes (Mawdesley-Thomas 1969, Hastein et al. tissue; (3) in the final, true ulcer stage the skin tissue 1978) have been associated with infections caused by was eroded and the muscle tissue was exposed (Wik- atypical A. salmonicida. Hyperplasia or proliferation of lund & Bylund 1993). Unfortunately, there are no re- the gills sometimes containing bacterial colonies has ports on histological studies of skin ulcers in flounder. been reported (Mawdesley-Thomas 1969, Morrison et Although there is accumulating evidence that atypi- al. 1984). In histological studies, the ulcers showed cal Aeromonas salmonicida is the aetiological agent of pathological changes with oedema, distortion of the CE, GUD and UDF, the subject is a matter of discus- scales, hyperaemia, haemorrhage, leucocytic infiltra- sion, and under certain circumstances other (oppor- tion and the presence of fibroblast-like cells often tunistic) pathogens can be isolated. In addition to A. causing granulomatous tissue in the dermis as well as salmonicida, the aetiology of CE, GUD and UDF has in the spleen and the kidney (Hastein et al. 1978, Mor- been associated with Aeromonas hydrophila, Vibrio rison et al. 1984). anguillarum, Flexibacter columnaris or opportunistic In non-salmonids there are few reports on signs of pathogens (Takahashi et al. 1975a, b, c, Nounou et al. pathological disease in internal organs of fish infected 1981, Sioutas et al. 1991, Ullrich 1992, Vethaak 1992, with atypical Aeromonas salmonicida. Congested and reviewed by Wiklund 1994b). However, some of these dark kidneys, spleens and gills were detected in gold- studies were inadequately reported or performed and, fish naturally infected with atypical A. salmonicida. hence, a successful isolation of atypical A. salmonicida Additionally, enlargement of the hepatocytes, degen- strains could not be ensured. Sioutas et al. (1991) and erative changes in the kidney, congestion and in some Vethaak (1992) did not specify the incubation time cases hyperplasia in the spleen were observed when examining ulcerated carp and flounder. Slow- (Mawdesley-Thomas 1969). Pathological signs of dis- growing strains of atypical A. salmonicida could have ease in the intestine (haemorrhage and hyperaemia) been overlooked due to a short incubation time. Vet- have been observed in captive sand-eels (Dalsgaard haak (1992) isolated A. salmonicida from flounder, but & Paulsen 1986) and farmed common wolffish Ana- it was not stated whether this was a typical or an atyp- rhichas lupus (Hellberg et al. 1996). Additionally, ical strain. Nounou et al. (1981) used selective or non- haemorrhages in the liver and in the musculature of enriched media in the isolation procedure, thus ham- infected sand-eels have been reported (Dalsgaard & pering the probable isolation of fastidious atypical A. Paulsen 1986). In infection experiments with atypical salmonicida from ulcerated flounder. A. salmonicida isolated from cod, degenerative changes, In American eel Anguilla rostrata, European eel accumulated leucocytes, and cyst formations were Anguilla anguilla, as well as Japanese eel Anguilla seen in the spleen and kidney of infected cod, while japonica, atypical Aeromonas salmonicida strains have in Atlantic salmon Salmo salar the injected bacteria been reported to cause severe necrosis, lesions of the caused no cellular host reaction except for the pres- skin, and tissue swelling on the head of affected speci- ence of necrotic muscle fibers at the injection site mens (Iida et al. 1984, Kitao et al. 1984, 1985, Ohtsuka (Morrison et al. 1984).In farmed wolffish, neither a cor- et al. 1984, Noga & Berkhoff 1990, Dalsgaard 199413). responding leucocyte reaction nor fibroblast encyst- The disease in eels has thus been referred to as 'head ment of atypical A. salmonicida was observed (Hell- ulcer disease' (Ohtsuka et al. 1984). A mild to severe, berg et al. 1996). However, Bulkley (1969), McCarthy primarily mononuclear infiltrate was observed in the (1975), Hdstein et al. (1978), Michel (1981), Wilson & Wiklund & Dalsgaard. Atypical Aeromonas salrnonicida: a revlew 5 7

Holliman (1994), and Jeney & Jeney (1995) reported ulcerations of rainbow trout Oncorhynchus mykiss that macroscopical signs of disease in internal organs caused by atypical A. salmonicida. However, tissue of fish (silver bream Blicca bjoerkna, carp, minnow, reaction or attempts at regeneration were not observed perch Perca fluviatilis, yellow bass Morone mississip- (Boomker et al. 1984). piensis) infected with atypical A. salmonicida were not Haemorrhages in the gills, liver, heart, intestine as observed, although Jeney & Jeney (1995) stated that a well as in the peritonea1 cavity of sea trout and lake slight fatty degeneration was sometimes visible in carp trout Salmo trutta m. lacustris have been reported affected by carp erythrodermatitis. According to Fijan (Ojala 1966, Wichardt 1983a). Boomker et al. (1984) (1972), the internal organs of carp affected with small observed the presence of a few gross changes in inter- ulcers appeared normal, but in the terminal stages nal organs, like slight splenomegaly, discoloration of fluid accumulation was found in the abdominal cavity the liver and enlargement of the kidney of rainbow and the internal organs were oedematous. In several trout infected with atypical A. salmonicida. Micro- studies no indication of pathological signs in internal scopically, the spleen and the liver were congested organs of non-salmonids infected with atypical A. sal- and in the kidney slight vasculitis and early degenera- monicida have been reported (Trust et al. 1980c, Whit- tion of tubular epithelium were encountered. Bacterial tington et al. 1987, Noga & Berkhoff 1990, Wiklund colonies were scarce and present only in sections of 1990, Wiklund & Bylund 1993, Pedersen et al. 1994, the skin. Wiklund & Dalsgaard 1995). The few signs of disease reported to be present in internal organs of fish indicate that atypical Aero- monas salmonicida seldom colonizes those organs. Salmonids This conclusion is in accordance with the fact that atypical A. salmonicida is only occasionally isolated In salmonids different atypical Aeromonas salmoni- from the internal organs of fish with an ulcerative cida strains have been associated with different patho- infection (see 'Isolation' and Table 5). Why the organ- logical gross signs. Wichardt (1983a) concluded that ism is retained at the site of the local skin lesion is the signs of disease in sea trout Salmo trutta m. trutta, unknown, but it might be a result of the effects of local which were caused by pigment-producing atypical A. inflammation and the host's immune response. Cer- salmonicida, more closely resembled those induced by tainly there are also differences in the invasive capac- achromogenic variants of atypical A, salmonicida than ity between different atypical isolates. More detailed those caused by typical strains. In contrast, Rintamaki studies on the infective mechanisms of different atypi- & Valtonen (1991) found pigment-producing atypical cal strains and corresponding immune response in the A. salmonicida caused signs of disease in salmonids host are needed. (mainly brown trout Salmo trutta m. fario and Atlantic salmon) that were similar to those caused by typical A. salmonicida but different from those caused by achro- MORTALITY mogenic variants of atypical A. salmonicida. The achromogenic strains caused mainly skin ulcerations Wild fish and other external or internal signs of disease were not detectable. According to Groman et al. (1992), both Epizootics, occasionally with high mortality, have A. salmonicida subsp. nova and typical A. salmonicida been observed in wild fish in connection with the iso- produced similar signs of disease in salmon, i.e. lation of atypical Aeromonas salrnonicida (Table 4). lethargy, aimless swimming, respiratory distress, fin However, for practical reasons the mortality rate for erosion and haemorrhagic cutaneous and muscular epizootics in wild fish has seldom been presented lesions. In some of the disease outbreaks in salmonids (McCarthy 1975). Additionally, the relationship be- caused by atypical A. salmonicida strains, ulcerations tween the mortality recorded and the isolated atypical in the infected fish were reported (Snieszko et al. 1950, A. salmonicida strains has not always been properly Paterson et al. 1980b, Boomker et al. 1984, Rintamaki & established (Bucke et al. 1979). High mortality in wild Valtonen 1991). Ulcerations were characteristically fish, suggested to be caused by atypical strains, has observed in disease outbreaks in brook trout Salveli- been described among yellow bass (Bulkley 1969). nus fontinalis associated with Hemophilus piscium silver bream (McCarthy 1975), minnow (Hastein et al. (Snieszko et al. 1950, Bullock et al. 1971), now classi- 1978), and perch (Michel 1981; strains subsequently fied as atypical A. salmonicida (Paterson et al. 1980a). identified as a likely atypical A. salmonicida, C. Michel Histologically, cellular infiltration, degenerative changes pers. comm.) (Table 4). In some of these cases, inade- in the epithelia1 and superficial muscle cells and con- quate nutrition (Bulkley 1969), transport of live fish siderable tissue destruction have been observed in (McCarthy 1975), or low water associated with spawn- 58 Dis Aquat Org 32: 49-69.1998

Table 4.Mortality rates in wild fish species and farmed salmon~dsassociated with atypical Aeronionas salrnonicida

Fish species Country Mortality Source

Wild fish species Silver bream UK 25 % morbidity McCarthy (1975) Perch UK >98% Bucke et al. (1979) Yellow bass USA Considerable Bulkley (1969) Minnow Norway Mass mortality HBstein et al. (1978) Perch France Mass mortality Michel (1981) Farmed salmonids Sea trout Sweden 60-65 % IVichardt (1983b) Atlantic salmon Canada -50% Paterson et al. (1980b) Rainbow trout South Africa 30 % Boomker et al. (1984) Atlantic salmon Canada >25% Groman et al. (1992) Different species Iceland 15-25% Gudmundsdottir et al. (1995) Sea trout Finland 2-20% Rintamaki & Valtonen (1991) Salmon Faroe Islands 7% Dalsgaard & Mortensen (1988)

ing season (Hbstein et al. 1978), which all may cause that the mortality in carp populations was caused by stressful conditions for the fish, were suggested as the invasion of opportunistic pathogens, facilitated by the primary cause of the infection with atypical A. salmoni- immunosuppressive substances released by atypical cida and subsequent heavy mortality observed. In A. salmonicida (Evenberg et al. 1986, Pourreau et al. some studies of ulcerated fish, mortality in the affected 1986, Sovenyi et al. 1988). population has not been reported (Wiklund 1990, Wik- lund & Bylund 1993, Wiklund & Dalsgaard 1995). How- ever, low mortality in a fish population, whatever the ISOLATION reason, will most probably remain undetected. Low numbers of weak and moribund fish will be eliminated While the isolation of typical Aeromonas salmonicida due to predation by fish, birds and mammals. is mostly a standard procedure and the cells readily grow on basic isolation agar, the recovery of atypical A. salmonicida is more difficult. Fastidious, slow-growing Farmed fish strains of atypical A. salmonicida (Bullock et al. 1971, Ishiguro et al. 1986, Wiklund & Bylund 1991, Olivier In farmed fish high cumulative mortality has been 1992, Pedersen et al. 1994, Wiklund & Dalsgaard 1995) reported in several studies (Table 4). Wichardt (1983b) have been reported to require agar supplemented with reported very high mortality (60 to 65 %) in farmed sea blood or serum for isolation and further cultivation trout in one farm in Sweden due to atypical Aeromonas (Paterson et al. 1980b, Ishiguro et al. 1986, Olivier 1992, salmonicida infections during unfavourable environ- Wiklund et al. 1994, Wiklund & Dalsgaard 1995). Mc- mental conditions. In farmed Atlantic salmon in Canada, Carthy (1977) reported that tryptone soya agar was 50% cumulative mortality was observed in 1974 superior to blood-containing medium for the isolation of (Paterson et al. 1980b). In 1991 over $500 000 in poten- atypical as well as typical A. salrnonicida. However, this tial sales were lost during disease outbreaks caused by view was not confirmed by any comparative analysis, atypical A, salmonicida, which was considered to be and it represented merely an opinion based on labora- the major economic constraint to the commercial tory experience. Austin (1993) found that atypical salmonid culture in the area (Groman et al. 1992). In strains did grow on blood agar, but not on brain-heart Iceland atypical A. salmonicida infections in farmed infusion agar or tryptone soya agar at isolation, but sub- fish have caused losses equal to 15-25% of the total cultures grew well on all 3 media. It has been sug- slaughter production (Guamundsdottir et al. 1995). In gested that the incidence of atypical A. salmonicida as a general, however, the mortality due to atypical strains pathogen of different fish species may have been un- has generally stayed low although occasional mor- derestimated in studies using agar medium without tality up to 20% (Rintamaki & Valtonen 1991), 30% blood (Paterson et al. 1980b). (Boomker et al. 1984) and 35 % (calculated from Peder- Several studies show that atypical Aeromonas sal- sen et al. 1994) have been reported (Table 4). monicida is predominantly isolated from skin ulcers The mortality in carp caused by atypical Aeromonas and only occasionally from visceral organs of fish of salmonicida has been found to reach 25 % in pond cul- different species (Table 5). This has been observed in ture (Sovenyi 1986). However, it has been suggested investigations of ulcer disease of brook trout (Snieszko W~klund& Dalsgaard: Atyp~calAeromonas salmon~clda:a revlew 5 9

Table 5 Isolat~on(% of total number of flsh examined) of atypical Aeronionas salmonicjda from ulcerations and v~sceralorgans of d~fferentflsh specles nr atypical A salmonlclda isolated but exact number of ~solatesnot reported

F~shspecles Country No. of lsolation from lsoldt~onfrom Source

fish examined ulcerations ('%) v~scei-alorgans (C%)

Flounder F~nland 162 N~klund& Bylund (1901) ( hub UK 14 M ilson & Hollinian (1994) ( 'od Canada 22 Cornick et a1 (1984) Goldfish Japan, UK, USA 83 Elllot & Shotts (1080a) Flounder Denmark & southern 16 Wiklund & Dalsgaard (1995) Baltlc Sea Roach UK 7 5 Hubbert & Willlams (1980) A~ner~caneel USA 20 Noga & Berkhoff (1990) "Number of fish examined for infection in vlsceral organs = 31 et al. 1950), CE (Csaba et al. 1980a, Sovenyi et al. 1988, other bacteria, and/or growth suppression of A. sal- Austin 1993),GUD (Elliott & Shotts 1980a, Whittington monicida by contaminants may have contributed to the et al. 1987, Austin 1993), head ulcer disease of eel failure to isolate this pathogen (Bootsma et al. 1977, (Noga & Berkhoff 1990), ulcerations in perch (Michel Elliott & Shotts 1980a, Bullock et al. 1983, Humphrey & 1981), ulcerated cod kept in tanks (Cornick et al. Ashburner 1993). 1984), ulcerations in roach Rutilus rutilus (Hubbei-t & In conclusion it seems essential to perform bacterial Williams 1980, Austin 1993), and skin ulcerations in isolation on recently developed skin ulcerations as well different wild flatfish species like flounder, dab as on the internal organs of fish, using agar supple- Lirnanda limanda, plaice Pleuronectes platessa and mented with blood or serum, in order to make a proper turbot Scophtl~almusmaxjmus from the Baltic Sea and identification of the aetiologic agent of the skin ulcer the North Sea (Wiklund & Bylund 1991, Wiklund & disease, especially in wild fish. Dalsgaard 1995) (Table 5). In other studies isolation of atypical A. salrnonicida has successfully been achieved from internal organs of non-salmonid wild fish kept in PATHOGENICITY tanks or net-pens (Dalsgaard & Paulsen 1986, Traxler & Bell 1988). The pathogenicity of different atypical Aerornonas sal- In farmed fish, atypical Aer-ornonas salmonic~da is inonicida strains seems to be highly variable (Table 6). often readily isolated from internal organs as well as However, comparison of the virulence of the examined from ulcers. This has been shown for farmed turbot in strains is difficult due to the varying methods used in Denmark, where atypical A. salrnonlcida was isolated the different studies. The assessment of virulence has from external ulcers and kidneys of ulcerated speci- been performed on different fish species, and the mens (Pedersen et al. 1994). Elliott & Shotts (1980a) administration of the bacteria by different routes reported that atypical A. salmonicida was more preva- of challenge (e.g. intraperitoneal, intramuscular, sub- lent in kldneys of goldfish which had ulcerations in the cutaneous, bath and rubbing into disrupted skin); the late stage of developnlent than in goldfish with ulcers amount of administered bacteria has varied greatly, in the early or intermediate stage of development. and several studies did not report whether the strain In several studies it was observed that atypical tested was freshly isolated or passed on artificial media Ael-ornonas salmonicida could not be isolated from the (agar) once or several times before the challenge ex- ulcers of all examined ulcerated fish (Table 5). Addi- periment was performed. In some studies the injected tionally, atypical A. salmonicida was isolated mainly amount of bacteria was not reported (Bucke 1980, from lesions or ulcers in an early stage of development, Michel 1981, Sovenyi & Ruttkay 1986, Sovenyi et al. and more seldom from chronic, terminal or healing 1988). The age and the genetic strain of the fish (e.g. lesions (Elliott & Shotts 1980a, Whittington et al. 1987, carp) can also significantly affect the resistance against Wiklund & Dalsgaard 1995). Generally, in later devel- bath challenge with atypical A. salrnonicida (Wie- opmental stages the ulcers had often been invaded gertjes et al. 1993). Additionally, the nutritional status by opportunistic pathogens and contaminating water of the fish (e.g. Arctic char Salvelinus alpinus, carp, microorganisms such as motile Aeromonas sp., Pseudo- grayling Thymallus thymallus) and the feeding regime monas sp. and Saprolegnia sp. (Bootsma et al. 1977, have also been shown to affect the resistance to chal- Elliott & Shotts 1980a, Noga & Berkhoff 1990). The lenge with atypical A. salmonicida (Sovenyi & Ruttkay slow growth of atypical A. salrnonicida, overgrowth by 1986, Pylkko 1993). 60 Dis Aquat Org 32: 49-69, 1998

Table 6. Aeromonas salmonicida. Virulence of atyplcal A, salmonicida strains isolated from various fish species i.p.: intraperi- toneal; i.m.: intramuscular; s.c.:subcutaneous; MLD: minimum lethal dose

Host fish Challenged Challenge Injected amount Mortality or LDso Source species fish species mode of bactena (no. of dead fish/no, of infected)

Silver bream Rainbow trout i.p. 1.7 X ~~"MLD) Not reported McCarthy (1975) Minnow Minnow i.p. 6 X 10' 5/5 Hristein et al. (1978) Turbot Atlantic salmon 1.p. Pedersen et al. (1994) Wrasse Atlantic salmon i.p. Frerichs et al. (1992) Bath Frerichs et al. (1992) Sand eels Rainbow trout i.p. Dalsgaard & Paulsen (1986) Flounder Flounder i.p. Wiklund (l995b) Flounder Rainbow trout i.p. Wiklund (1995b) Flounder Roach i.p. Wiklund (1995b) Flounder Bleak i.p. Wiklund (1995b) Cod Atlantic salmon 1.m. Cornick at al. (1984) i.p. Cornick et al. (1984) Goldfish Goldfish Elliot & Shotts (1980b) Goldfish Goldfish Trust et a1 (1980~) C yprinid Goldfish i.m. Austin (1993) Cyprinid Rainbow trout i.m. Austin (1993) Wrasse Goldsinny wrasse i.p. 27 and 28/30 Gravningen et al. (1996) Japanese eel Japanese eel i.m. Ohtsuka et al. (1984) Pacific herring Pacific herring i.p. Traxler & Bell (1988) Goldfish Atlantic salmon i.p. Carson & Handlinger (1988) Goldfish Atlantic salmon 1.p. Whittington & Cuhs (1988) Goldfish Brown trout i.p. Whittington & Cullis (1988) Goldfish Brook trout i.p. Whittington & Cullis (1988) Goldfish Rainbow trout i.p. Whittington & Cullis (1988)

dLowest concentration of bacteria giving mortality; binjected bacteria not re-isolated; 'per 100 g fish; dcalculated 10 d LDS,

In some studies the virulence of isolated strains was Evenberg et al. (1986) concluded that the pathogen- tested on several fish species (Rucke 1980, Michel esis of CE caused by atypical Aeromonas salmonicida 1981, Whittington & Cullis 1988, Austin 1993, Wiklund is characterised by a state of immune suppression in 1995b). Some of the examined strains were pathogenic the infected fish. This may explain why opportunistic only for the original host and not for other fish species pathogens such as Aeromonas hydrophila, Pseudo- challenged (Michel 1981, Wiklund 1995b), while in monas sp., Vibno sp. and Vibrio anguillarum can be other studies the examined strain was pathogenic for isolated from internal organs of moribund, seriously several fish species (Bucke 1980, Whittington & Cullis ulcerated fish or from fish injected with atypical strains 1988, Austin 1993). Michel (1981) found that an atypi- (Csaba et al. 1980a, Cornick et al. 1984, Evenberg et cal strain isolated from perch was non-pathogenic to al. 1986, Wiklund 1995b, Gravningen et al. 1996). The carp, possibly pathogenic to rainbow trout and patho- cause of a possible immune suppression in fish in- genic to perch when injected intramuscularly. Wiklund fected with atypical A, salmonicida could be strain spe- (1995b) reported an atypical A. salmonicida strain iso- cific, because in other experimental studies invasion of lated from flounder to be weakly pathogenic for floun- opportunistic pathogens has not been observed in der and non-pathogenic for rainbow trout. Addition- injected fish (Dalsgaard & Paulsen 1986). In infection ally, mortality among challenged roach and bleak experiments on carp, opportunistic pathogens (previ- Alburnus alburnr~soccurred, but the injected pathogen ously isolated from ulcers) failed to produce ulcers in could not be re-isolated; instead other bacteria in- injected fish (Csaba et al. 1980a, Evenberg et al. 1986). cluding Aeromonas sp., Pseudomonas sp. and Entero- In another study, A. hydrophila, isolated from ulcer- bacteria were isolated. It was suggested that the ated carp, produced clinical and pathological disease injected atypical A. salmonicida strain caused an signs identical to CE, but the amount of bacteria immunosuppressive effect on the challenged fish, injected or rubbed into scarified skin of carp was rather allowing opportunistic pathogens to invade the fish high (3.4 X log bacteria) (Sioutas et al. 1991), making (Wiklund 1995b). the experimental evidence questionable. Elliott & Wiklund & Dalsgaard: Atypical Aeromonas salmonidda: a review 6 1

Shotts (1980b) reported that an anaerogenic member that the invading capacity of the highly pathogenic of the A. hydrophila complex, isolated from ulcers of (at i.p. injection) strain tested through the water, into diseased goldfish, did not produce ulcerations or salmonids, was rather limited, although Whittington & mortality in bath challenged goldfish at a concentra- Cullis (1988) concluded that the degree of bacterial tion of 7.2 X 106 colony-forming units (CFU) ml-'. contamination of atypical A. salmonicida of the tank In infection experiments Bucke (1980) found that a environment was probably low and that the low con- weakly pigment-producing strain of Aeromonas sal- centrations were infective. Unfortunately, no studies monicida was more virulent for several fish species on the concentration of atypical A. salmonicida in the (brown trout, carp, goldfish, roach, rudd Scardinius water were done, so the above conclusion is based erythrophthalmus and perch) than a non-pigmented more on assumption than on fact. Additionally, in bath strain of A. salmonicida. Unfortunately, the amount of experiments, injection (bacterial concentrations: 2.0 X bacteria injected was not reported. The different 10' to 2.0 X 106 CFU ml-') with the same highly patho- numbers of cells of the 2 strains injected into the fish genic atypical strain as used for i.p. resulted in low could explain the difference in the virulence observed. mortality with challenged Atlantic salmon and rain- Additionally, it was reported that a weakly pigmented bow trout, but high mortality with brown trout and variant of A. salmonicida could be experimentally brook trout. The CO-habitation and bath experiments transferred by CO-habitation from infected brown trout probably give a better estimate of the virulence and to several fish species such as brown trout, carp, gold- invasive capacity of the tested pathogen than the injec- fish and roach (Bucke 1980). tion experiments. In a similar experiment done with Some strains of atypical Aeromonas salmonicida iso- another atypical strain isolated from ulcerated goldfish lated from non-saln~onids were reported as extremely high mortality was obtained with i.p. injection (LDS, = virulent (Ohtsuka et al. 1984, Carson & Handlinger 3 CFU) as well as bath challenge (8.0 X 10' CFU ml-l) 1988, Traxler & Bell 1988, Whittington & Cullis 1988), of Atlantic salmon (Carson & Handlinger 1988). Whit- with calculated 10 d LDjo values as low as 7.4 X 10-3 tington & Cullis (1988) found that an atypical strain CFU by intraperitoneal injection (i.p.) (Whittington & isolated from goldfish could establish a carrier state in Cullis 1988). The LDS0 value in this study was esti- i.p.-injected Atlantic salmon. These authors concluded mated, by regression analysis, to be the dose for which that outbreaks of furunculosis in salmonid fish will the median lifetime of challenged fish was 10 d. As the occur if the tested strain of atypical A. salmonicida is atypical strain used in the experiments autoaggluti- transmitted from goldfish to salmonids. nated in broth, 1 CFU consisted of a clump of bacteria, Although atypical Aeromonas salmonicida has been which accounts for the LDso values that were less than isolated from several salmonid fish species (Table 3), 1 unit (Whittington & Cullis 1988). Other examined there are surprisingly few reports on the virulence of strains seemed to be totally non-virulent as determined the isolated strains. Olivier (1992) reported that strains in injection experiments (Pedersen et al. 1994, Mc- (subsp. nova) isolated from salmonids in Newfound- Carthy 1975) (Table 6). land, Canada, were highly pathogenic (LDS, < 100 bac- In many studies the virulence of atypical strains iso- teria) for juvenile Atlantic salmon and brook trout. lated from non-salmonid fish was assessed on fish used Unfortunately, several virulence and transmission for farming, mainly rainbow trout or Atlantic salmon, studies of atypical Aeromonas salmonicida strains while the virulence for the host fish species was not were done by injecting the pathogen into the fish determined (Table 6). The explanation for this could be (Table 6), which does not give a good picture of the the need to gain knowledge about the possible spread infection capacities of the examined bacteria. The of the disease from wild fish to the more economically technique using application of bacteria into scarified important farmed fish. Tested atypical Aeromonas sal- skin has been applied mainly in the examination of CE monicida strains isolated from non-salmonids seem to (Bootsma et al. 1977, Csaba et al. 1980a, Gayer et al. be non-virulent or low-virulent for salmonids (Table 6), 1980, Mirle et al. 1986). However, the amount of bac- except for strains isolated from goldfish in Australia, teria applied to the scarified skin is difficult to control which have proved to be extremely virulent by i.p. using this method, and observed differences in viru- injection of different salmonids (Carson & Handlinger lence (40 to 100%) of examined strains (Bootsma et al. 1988, Whittington & CulLis 1988). However, in experi- 1977) could be explained by the use of different con- ments performed simultaneously with i.p. injection, centrations of the organisms tested. In the analysis of the atypical strain was not transmitted via water from GUD and UDF, abrasion of the shn and bathing in the injected fish to Atlantic salmon and rainbow trout a bacterial suspension of A. salmonicida has been kept in the same tanks as the infected fish (within-tank performed in different studies. The concentration of control or CO-habitation) but was transmitted to a low bacteria in the suspension has often been rather high number of brown trout and brook trout. This indicates (2 X 105 to 4.3 X 107 CFU ml-l) (Elhott & Shotts 1980b, 62 Dis Aquat Org 32: 49-69, 1998

Carson & Handlinger 1988, Whittington & Cullis 1988, atypical strains were detected, while the examined Wiklund 1995b). A method using micro-injection, with typical strains formed a homogenous, single protease Hamilton syringe, of a small amount of bacteria has group. The results clearly indicate that with respect to been used as a compromise between scarification extracellular proteases the atypical strains also form a technique and immersion route (Evenberg et al. 1986, very heterogeneous group. Gudmundsdottir (1996) Evenberg et al. 1988). However, in order to obtain an concluded that exoprotease production appeared to be appropriate description of the virulence of atypical strongly associated with geographical location of the strains, future investigations on this issue need to use host rather than the host fish species itself. other methods like CO-habitation, which so far has A relationship between the presence of an extracel- been employed only in a few studies on the patho- lular A-layer, external to the cell wall of atypical and genicity of atypical A. salmonicida (Bucke 1980, Whit- typical Aeromonas salmonicida, and virulence has tington & Cullis 1988). been suggested (Kay et al. 1981, Evenberg et al. 1988, Austin & Austin 1993). However, the close association between virulence and the presence of A-layer in VIRULENCE FACTORS typical strains is still being debated, and there are reports of non-virulent isolates possessing an A-layer Present knowledge about virulence factors in atypi- and virulent isolates with no detectable A-layer (Ellis cal strains is rather limited compared to knowledge et al. 1988, Olivier 1990, Austin & Austin 1993). A-layer- about typical strains of Aeromonas salmonicida. The possessing as well as A-layer-deficient atypical A. presence of extracellular products (ECPs) including salmonicida strains have been isolated from different proteases, A-layer of the cell wall, and iron-uptake fish species (Trust et al. 1980a, Kay et al. 1981, Even- mechanisms of atypical strains have been described in berg & Lugtenberg 1982, Evenberg et al. 1982, Chart several studies. ECPs from atypical strains have been et al. 1984, Kay et al. 1984, Pedersen et al. 1994, examined by Pol et al. (1980), Hastings & Ellis (1985), Wiklund et al. 1994). Detailed investigations on the Evenberg et al. (1988), Gudmundsdottir et al. 1990, virulence of A-layer-possessing and -deficient atypical and Gudrnundsdottir (1996). Pol et al. (1980) reported strains have not been performed, but as previously that crude ECP of an atypical A. salmonicida strain was indicated the virulence of different atypical strains can lethal for carp, but the nature of the toxin(s) was not be host-specific and the virulence of a certain strain determined. They concluded that ulcers occurring in has to be assessed on the same fish species from which connection with CE are partly or exclusively caused by the examined strain was originally isolated (Table 6). a bacterial toxin produced by atypical A. salmonicida. Bacteria have an absolute requirement for iron, and Evenberg et al. (1988) found that the lethality of cell- a bacterial pathogen must possess high-affinity iron- free culture supernatants of atypical A. salmonicida for uptake mechanisms which can compete with the iron- carp varied significantly depending on the composition binding proteins (such as transferrin) in the serum and of the growth medium and incubation time. The mor- extracellular fluids of the host. Such mechanisms tality of the challenged fish ranged from zero (over include the production of siderophores and the induc- 10 d) to death within 48 h. Hastings & Ellis (1985) ex- tion of transferrin-binding proteins under conditions of amined an atypical strain from Iceland which did iron restriction. Siderophore production has not been not produce any detectable haemolysin or gelatinase observed in atypical strains (Chart & Trust 1983, Hirst and, in contrast to typical strains, had caseinase prop- et al. 1991), and iron-uptake has been suggested to be erties comparable to those of a metallo-protease. Sub- performed by a probable proteolytic degradation of sequently, Gudmundsdottir et al. (1990) reported that a transferrin bound iron by an extracellular metallo- metallo-protease with a molecular weight of approxi- protease (Hirst & Ellis 1996). A probable correlation mately 20 kDa was the major extracellular lethal toxin between established protease groups (Gudmundsdot- of atypical strains isolated from salmonids in Iceland. tir 1996) and uptake of iron might explain the large The 24 h value of the protease was 0.03 pg protein variations in virulence observed in different atypical g-' fish. This protease was different from the 2 extra- strains (Table 6). However, future research will hope- cellular proteases (P1 and P2) described from fully resolve this issue. The utilisation of haem-bound A. salmonicida subsp. salmonicida. In contrast, strains sources of iron have been reported to be A-layer isolated from cyprinids (goldfish, carp, and rudd) did dependent both In atyplcal and typical Aeromonas not produce this metallo-protease (Gudmundsdottir et salmonicida (Hirst et al. 1994). Also, the utilisation of al. 1990). In a recent study, 25 atypical A. salmonicida haem-bound iron by atypical A. salmonicida indicated strains showed significant differences in the produc- that the mechanism of haem utilisation was not tion of extracellular proteases (Gudmundsdottir 1996). siderophore-mediated, due to the lack of siderophores Five different protease groups among the examined in atypical strains (Hirst et al. 1991). Wiklund & Dalsgaard: AtypicalAeromonas salrnonjcida: a review 63

ECOLOGY CONTROL

Compared to typical Aeromonas salmonicjda The prophylactic and therapeutic methods available (Michel & Dubois-Darnaudpeys 1980, Allen-Austin et for the control of diseases caused by typical Aero- al. 1984, Rose et al. 1990, Morgan et al. 1991, Effendi monas salmonicida have been reviewed by Austin & & Austin 1994, Husevbg 1994, Ferguson et al. 1995), Austin (1993). For diseases caused by atypical A. sal- there is limited information available about the eco- moniczda less information about control is available. It logy, spread and survival of atypical strains in water appears that potentiated sulphonamide, chlorampheni- (Evelyn 1971, Wiklund 1995a). Evelyn (1971) found col, neomycin, nitrofurantoin and oxytetracycline are that an atypical strain isolated from sablefish survived generally effective against these pathogens (Bootsma better in sea water than in fresh water (distilled or et al. 1977, Csaba et al. 1980a, Gayer et al. 1980, Bohm tap water). The addition of peptone to the sea water et al. 1986, Groman et al. 1992, Pedersen et al. 1994, extended the survival time significantly. Wiklund Jeney & Jeney 1995). (1995a) examined the survival of atypical A. salrnoni- The development of resistance to antibiotics among cida (isolated from ulcerated flounder from brackish atypical strains has been observed by Hirvela-Koski water) under laboratory conditions in glass bottles et al. (1994). who found resistance to sulfonamides. (microcosms) containing sterilised water and sedi- Strains isolated from turbot (Pedersen et al. 1994) were ment. In sterilised water the examined strains showed resistant to trimethoprim but sensitive to potentiated better survival at 4OC than at 15°C. In contrast, in sulphonamide. Transferable drug resistance has been water and sediment slightly increased survival was known to occur in typical Aeromonas salmonicida observed at 15OC compared to 4'C. The strains tested since 1959, when Aoki et al. (1971) demonstrated the showed the highest survival in brackish water (S = presence of R-factors. Sandaa & Enger (1996) demon- 6.4 + 0.5%0) compared to fresh water or salt water strated transfer of a naturally occurring plasmid (S = 30%,,). Wiklund (1995a) concluded that bacteria (pRASl), encoding multiple drug resistance, from shed from ulcers of flounder may survive in the bot- atypical A. salmonicida to a variety of marine bacteria. tom sediment of brackish water environments in Little information is available about immunological excess of 60 d. The sediment can thus act as a reser- protection of fish against infection with atypical voir for the pathogen, facilitating the spread of the Aeromonas salmonicida. Evenberg et al. (1986) and infection, although the question of whether or not the Pourreau et al. (1986) showed that carp infected with virulence of the pathogen was preserved was not atypical A. salmonicida had a suppressed humoral examined (Wiklund 1995a). Although the atypical immune response, while an experimental vaccine con- strains isolated from ulcerated flounder have been sisting of the same strain, demonstrated that ECP is reported to be fastidious, requiring serum or blood important for protection (Evenberg et al. 1988). Daly when cultivated on artificial agar plates (Wiklund & et al. (1994) found that carp have memory formation Dalsgaard 1995), their survival in nutrient-limited in response to previous infection with atypical A. conditions (sediment and brackish water) was clearly salmonicida and that the protection lasted for at extended. However, these survival studies were done least 5 mo. Vaccination against atypical 'furunculosis' in sterilised water in microcosms, without any ex- (caused by atypical A. salmonicida) in reared Atlantic change of nutrients and waste products or interaction salmon in Newfoundland has not successfully elimi- between the bacteiia tested and other bacteria or nated carriers or alleviated disease in fresh or sea protozoans. Therefore, caution must be used in the water (Groman et al. 1992). Hirst & Ellis (1994) found interpretation of the results, and the information that iron-regulated outer membrane proteins (IROMPs) obtained does not necessary reflect the behaviour of of A. salrnonicida represent protective antigens, which the pathogen in the natural aquatic environment have an important role in the formulation of a success- (Austin & Austin 1993). ful vaccine against both typical and atypical strains of The few data presently available about the survival A. salmonicida. of atypical strains are based only on laboratory results. Farmed salmonids have been vaccinated with good For a proper evaluation of the possible spread of results against atypical 'furunculosis' in Iceland with atypical strains from infected populations to uninfected an autogenous bacterin since 1992. Field studies indi- ones more information is needed, especially on sur- cated some cross-protection of the autogenous bac- vival of different atypical strains in natural habitats. terin of Aeromonas salrnonicida subsp. achrornogenes Additionally, more knowledge is needed on the effect against classical furunculosis (Gudmundsdottir et al. of prolonged survival and starvation in water habitats 1996). However, in a recent study protection against on the maintenance of the virulence of the surviving classical furunculosis was not observed in fish vacci- cells. nated with an A. salrnonicida subsp. achrornogenes 64 Dis Aquat Org 32: 49-69, 1998

bacterin. The fish were kept in tanks under laboratory the pathogen in water and sediment is needed, and it conditions. The authors suggested that a constant ex- will be necessary to assess the capacity of the bacteria posure to the bacterium under field conditions boosts to survive in a non-culturable state and, in particular, and improves the immune response (Gudmundsdottir to determine whether the virulence capability of the & Gudmundsdottir 1997). Gudmundsdottir & Mag- starving cells changes in aquatic environments. The nadottir (1997) have recently assessed the protective bacteria have been isolated from clinically healthy fish, role of cell-associated and extracellular antigens of A. and it seems most likely that the fish have to be sub- salmonicida subsp. achromogenes. The results indi- jected to stressful conditions before the disease breaks cate that a specific humoral immune response evoked out. At present, it is unknown whether the bacteria in Atlantic salmon by a 20 kDa extracellular metallo- constitute a part of the normal flora of the fish, whether caseinase, AsaP1, is important in protection against the pathogen is transmitted for long distances through experimental infection of A. salmonicida subsp. achro- the water, or whether transfer occurs from fish to fish, mogenes. Jones et al. (1996) observed that vaccination during spawning for example. against typical furunculosis protects against atypical The biochemical identification of Aeromonas sal- A. salmonicida in salmonids and cyprinid fish. These monicida to species level can be done, remembering authors suggested that antigens capable of eliciting that several of the atypical strains are fastidious and protective immunity are conserved among biochemi- they need special growth media. In addition, strains of cally diverse strains of A. salmonicida. A, salmonicida with divergent characters are isolated both within the typical and atypical groups. Until the last decade the subspecies proposed by Schubert CONCLUSIONS (1967) were easily distinguished by a limited number of biochemical characters. This classification was This review on atypical Aeromonas salmonicida de- accepted by Bergey's Manual of Systematic Bac- monstrates that, despite the large amount of informa- teriology (Popoff 1984). The proposal of McCarthy & tion available about this important fish pathogen, sub- Roberts (1980) that A. salmonicida should be divided stantial gaps remain in our knowledge not only about into different subspecies based on epizootiological the non-specific factors influencing host specificity, vir- criteria, with most of the atypical strains included into ulence determinants, and survival of the pathogen in the subspecies nova, in hindsight seems to be too the environment but also about specific factors related simple a solution. However, the present situation with to the atypical strains, including the relevance of these 2 completely different systematic groups of subspecies strains to farmed fish, their taxonomy, and the question is impractical and taxonomically inappropriate. There of whether individual strains spread and, if so, how far. is an urgent need to clarify the taxonomic position of Atypical Aeromonas salmonicida seems to infect a atypical strains, and identification of the different sub- large number of fish species worldwide. The disease species or groups of atypical A. salmonicida using mol- problems have shown themselves to be more conspic- ecular techniques will be a priority for future work. uous in farmed fish than in wild fish, where generally With respect to fish health, the atypical strains are be- only small numbers of fish are diagnosed with ulcera- coming more and more important, resulting in an in- tions associated with atypical A. salmonicida. The crease in the demand for a valid definition of these infection studies carried out so far show that the viru- pathogens and also for improved prevention and con- lence of atypical A. salmonicida varies according to the trol procedures. Fish populations infected with atypical host species. These observations indicate, therefore, Aeromonas salmonicida have been transferred to geo- that there might be host species variation in suscepti- graphical areas (countries) that are considered free bility of different strains of atypical A. salmonicida. of (classical) furunculosis (Whittington & Cullis 1988), Some of the atypical strains isolated from wild fish where spread of infection occurred because of unsuc- adversely affect salmonids and therefore may repre- cessful attempts at treating the disease and absence sent a significant risk to farmed fish, both salmonids of legislation. Should we accept the trading of fish in- and non-salmonids. At present, there is a trend on fected with certain biotypes of atypical A. salmonicida, introducing new non-salmonid fish species for farming while infection with others is prohibited? Additionally, purpose and the susceptibility of these fish species to can a geographical area or country be considered free atypical A. salmonicida has to be examined. Addition- of A. salmonicida if atypical strains have been isolated ally, we need more information about the possible from fish in that area. Certainly, more information, re- change of host specificity of atypical strains. search and discussion are needed on these issues! Studies on the role of the environment as a reservoir This review will hopefully provide a basis of infor- of atypical Aeromonas salmonicida would seem to be mation that can be useful in future studies on the of particular importance. More work on the survlval of species Aeromonas salmonicida. Wiklund & Dalsgaard: Atypical Aero~nonassalnionicida: a rev~ew 65

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Editorial responsibility: David Bruno, Submitted: April 4, 1997; Accepted: November 14, 1997 Aberdeen, Scotland, UK Proofs received from author(s): February 10, 1998