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B12-bgICEL. AGR. SCI. 12, 1998: 61–72 Infections by atypical strains of the bacterium
Institute for Experimental Pathology, University of Iceland, Keldur, IS-112 Reykjavík Infections due to atypical strains of the Gram-negative bacterium Aeromonas salmonicida cause atypicalfurunculosis and related diseases of both feral and cultivated fish stocks in freshwater and marineenvironment. More than 20 farmed and 30 wild fish species have been reported to harbor atypical A.
salmonicida. The isolated strains are found to be a heterogeneous group as regards many phenotypic andgenotypic characteristics. The clinical and pathological features of infection are different as manyfactors are involved, e.g. various hosts, strains and environment. Infections of fish in the temperateregions of the Northern hemisphere are most frequently reported, although disease problems have alsooccurred in other parts of the world as Australia, the Mediterranean and Chile. In Iceland atypicalfurunculosis has been the main bacterial disease in the salmonid farming industry. As diseases caused byatypical strains are of emerging importance worldwide, the prospects of their control by vaccinationneed to be considered. Currently all commercially available A. salmonicida vaccines are produced fromtypical A. salmonicida strains for prevention of classical furunculosis in salmonids. There is, however,evidence of cross protection against atypical furunculosis in Atlantic salmon vaccinated with commer-cial oil based furunculosis vaccine. Icelandic fish farmers have since 1992 vaccinated salmonids byinjection with an autogenous bacterin produced by a commercial vaccine producer against atypical A.
salmonicida and recently halibut farmers in Norway have started to use an autogenous injection vaccineagainst atypical furunculosis.
Key words: Aeromonas salmonicida, atypical strains, bacteria, fish, fish pathogens, furunculosis, infec-tion, skin ulcers, vaccination.
YFIRLITSýkingar atýpískra stofna bakteríunnar Aeromonas salmonicida Sýkingar atýpískra stofna Gram-neikvæðu bakteríunnar Aeromonas salmonicida valda kýlaveikibróðurog hliðstæðum sjúkdómum í villtum og ræktuðum fiski, bæði í fersku vatni og sjó. Bakterían hefur veriðeinangruð úr yfir 20 tegundum eldisfisks og meira en 30 tegundum af villtum fiski. Rannsóknir á stofna-söfnum hafa leitt í ljós töluverðan breytileika á svipfari og erfðaeiginleikum atýpískra A. salmonicidastofna. Sjúkdómseinkenni eru breytileg og hafa ýmsir þættir þar áhrif, s.s. mismunandi hýslar, stofnar ogumhverfi. Sjúkdómsvandamál eru algengust á norðlægum slóðum, en þó hafa komið upp vandamál íöðrum heimshlutum eins og í Ástralíu, Miðjarðarhafslöndum og Chile. Kýlaveikibróðir er sá bakteríu-sjúkdómur sem mestum skaða hefur valdið í íslensku fiskeldi. Þar sem vandamál vegna sýkinga af völdumatýpískra A. salmonicida stofna hafa aukist á veraldarvísu er nauðsynlegt að íhuga möguleika á notkunbóluefna sem sjúkdómsvarnar. Rannsóknir hafa leitt í ljós töluverðan mun á sýkiþáttum mismunandiA. salmonicida stofna. Týpískir stofnar eru einsleitur hópur hvað varðar framleiðslu ensíma sem eru virkí sýkingu bakteríunnar, en meðal atýpískra stofna er mikill breytileiki. Sem stendur eru öll markaðsettA. salmonicida bóluefni framleidd til varnar klassískri kýlaveiki í laxfiskum. Til eru niðurstöður sem sýnakrossvörn gegn kýlaveikibróður í laxi sem var bólusettur gegn klassískri kýlaveiki. Íslenskir fiskeldis- menn hafa frá 1992 bólusett laxfiska gegn kýlaveikibróður með sérlöguðu (autogenous) sprautubóluefniog lúðubændur í Noregi hófu nýlega bólusetningu með sérlöguðu sprautubóluefni gegn sýkingu atýpískraA. salmonicida stofna. Notkun sérlagaðra bóluefna takmarkast við það svæði eða þá stöð sem sá bakteríustofnsem notaður er við gerð bóluefnisins var einangraður á.
The first definitive isolation of the Gram-nega- occur in both cultivated and feral fish stocks tive fish pathogenic bacterium Aeromonas sal- and more than 20 farmed and 30 wild fish spe- monicida was from brown trout (Salmo trutta) cies have been reported to harbor atypical A. salmonicida (Wiklund and Dalsgaard, 1998).
more than a century ago. As the bacterium hasbeen a continious tread to the salmonid farm- ing industry it has presumably been studied Like typical strains of A. salmonicida, atypi- cal variants are described as a non-motile, oxi- Currently the taxonomic status of A. salmon- dase-positive, fermentative, facultative anaero- icida is within the family Aeromonadaceae, bic, Gram-negative rods. Coccoid forms oc- in the genus Aeromonas (Colwell et al., 1986; cur frequently and staining has a tendency to Anonymous, 1992). Four subspecies of A. sal- be bipolar. The colonies on agar are after 2–5 monicida have been described, i.e. ssp. salmon- days incubation circular, raised, friable and of icida, achromogenes, masoucida and smithia a variable size. Many strains produce a water- (Holt et al., 1994). A. salmonicida ssp. salmon- soluble brown pigment when grown on tryptone- icida, the causative agent of classical furun- containing media (Popoff, 1984). The optimal culosis of salmonids, is called typical, but other growth temperature is 22–25°C and most strains strains atypical. Although three subspecies do not grow at 37°C. However, there is a mo- within the atypicals have been described, some tile biogroup that does grow at this tempera- authors have suggested different delineation ture. Furthermore, atypical as well as typical and new isolates that do not fit into the exist- A. salmonicida that lack the oxidase reaction ing classification are frequently reported. There- have been reported. This has made the identi- fore, an atypical strain can only be defined as fication of the bacteria more complicated. Se- a strain that does not fit into the existing clas- rological differences have not been detected sification of A. salmonicida ssp. salmonicida.
among fresh isolates of A. salmonicida, which A. salmonicida ssp. salmonicida has been makes serological methods feasible for identi- described as a homogeneous taxon, with re- fication to the species level (Bernoth, 1997).
spect to biochemical and genotypic charac- Atypical isolates are characterized by bio- teristics (Austin and Adams, 1996). On the chemical properties differing from those de- contrary the group of atypical A. salmonicida scribed for A. salmonicida ssp. salmonicida.
strains consists of isolates showing a large These most often include reduced or slow pig- variety of biochemical, molecular and viru- mentation, slow growth, nutritional fastidious- lence characteristics (Austin et al., 1998).
ness (often requiring blood products), growth One of the earliest indications of the exist- at elevated temperatures or oxidase-negativ- ence of aberrant A. salmonicida strains dates ity. Although pigment production is usually back to 1963 when Smith described atypical associated with A. salmonicida ssp. salmon- non-pigmented strains (Smith, 1963). With the icida, there have been some reports of typical enormous expansion in aquaculture and culti- isolates that do not produce pigment (Wik- vation of more fish species the economic im- lund and Dalsgaard, 1998). Phenotypic char- portance of infections due to atypical A. salmon- acteristics, which can be used for distinguish- icida has concurrently increased. Epizootics ing subspecies and atypical strains of A. sal- Table 1. Characteristics for differentiation between Aeromonas salmonicida ssp. salmonicida and atyp-
ical A. salmonicida. +, positive reaction (≥80%); –, negative reaction (≤20%); d, different results (21–
79%); n.d, no data available; R, resistant; S, sensitive.
1. tafla. Eiginleikar til að greina á milli Aeromonas salmonicida ssp. salmonicida og atýpiskra A. sal-
monicida baktería. +, jákvæð svörun (≥80%); –, neikvæð svörun (≤20%); d, mismunandi niðurstaða
(21–79%); n.d, engar niðurstöður fundust; R, þolin; S, næm.
Data are from the following publications: Austin et al., 1989; Olivier, 1992; Holt et al., 1994; Wiklundet al., 1994; Austin et al., 1998; Wiklund and Dalsgaard, 1998. The data are obtained by different labo-ratories, methods and times of incubation (from 2–14 days at 20–25°C)—Eftirfarandi heimildir eru fyrirniðurstöðum: Austin o.fl., 1989; Olivier, 1992; Holt o.fl., 1994; Wiklund o.fl., 1994; Austin o.fl., 1998;Wiklund og Dalsgaard, 1998. Niðurstöðurnar eru unnar af mismunandi rannsóknarhópum, sem notuðumismunandi aðferðir og ræktunartíma (frá 2–14 dagar við 20–25°C).
monicida are listed in Table 1. The data pre- also been isolated from fish in Australia and sented in Table 1 have, however, to be inter- in the Mediterranean (Wiklund and Dalsgaard, preted with caution, as it has been shown that 1998). Since 1995 infections by atypical A. large discrepancies occur in biochemical iden- salmonicida have also caused disease prob- tification of atypical strains obtained in vari- lems in cultivated salmon in the south of Chile, ous laboratories (Dalsgaard et al., 1998).
were the salmon production is very high. In Atypical A. salmonicida strains have been this area fish farms have been using salmon isolated from a wide range of fish species in eggs imported from countries where atypical freshwater, brackish water as well as in seawater A. salmonicida is endemic (Sandra Bravo, per- The geographical distribution of reported iso- lations indicate that atypical strains mainly in- The term furunculosis is commonly used for fect in the temperate regions of the northern all diseases of teleost fish caused by A. salmon- hemisphere, that is Canada, USA, Japan, and icida. However, this is an inappropriate desig- central and northern Europe, including the nation insofar as the classical necrotic lesions Nordic countries. However, atypical strains have or ulcers that develop are not typical abscesses as seen in mammals. In some regions infec- merlangus), four bearded rockling (Enchely- tions by typical A. salmonicida strains may opus cimbrius), bream (Abramis brama), dace overshadow those of atypical strains, espe- (Leuciscus cephalus), minnows (Phoxinus cially where pigment producing atypical strains phoxinus), perch (Perca fluviatilis), roach are involved (Austin and Austin, 1993).
(Rutilus rutilus) and pike (Esox lucius) (Wik- The clinical and pathological features of infections by atypical A. salmonicida strains In Iceland atypical furunculosis caused by can vary. Many factors are involved, e.g. en- A. salmonicida ssp. achromogenes was first vironmental factors, virulence properties of diagnosed in 1980, in the early days of the the respective bacterium and different host re- salmonid farming industry. Ever since it has sponses. This makes the diagnosis difficult as been a threat to the fish farming industry. The the atypical strains are a heterogeneous group disease problems have been most severe in that infect various fish species both in fresh- fish reared in tanks with brackish water (salin- water and marine environment. Atypical A. ity between 0.3 to 2%), where accumulative salmonicida strains are the cause of ulcera- mortality as high as 30% has been recorded.
tive and systemic infections in a wide variety The disease is frequently diagnosed in feral of fish including many economically impor- salmonids and outbreaks of the disease have tant species as, salmonids, carp, goldfish, cod, also occurred in captive cod of wild origin eel, turbot, flounder, halibut and many others.
reared in land based tanks. The susceptibility The best known diseases include goldfish ul- of halibut to the bacterium has been shown by cerative disease, carp erythrodermatitis, ulcer an experimental infection. The bacterium has disease of flounder, eel and salmonids and atypi- also been isolated from several wild fish spe- cal furunculosis of salmonids and several other cies in the Icelandic waters (Gudmundsdóttir, fish species. Apparently atypical strains are involved in more disease outbreaks in fish thanwas previously suspected (Wiklund and Dals- The clinical presentation of atypical Infections by atypical A. salmonicida have Atypical A. salmonicida infections associated caused serious problems in the farming of with disease outbreaks in fish (e.g. atypical salmonids in Canada and all the Nordic coun- furunculosis of salmonids, cod and common tries, with the exception of Denmark. In these wolffish) can be manifested, similar to furun- countries problems have also occurred in the culosis, as loss of appetite with darkening in farming of non-salmonids as cod (Gadus colour and increased mortality. External clinical morhua), halibut (Hippoglossus hippoglossus), signs often include other features of an acute common wolffish (Anarhichas lupus), spot- septicaemia like haemorrhage at fin bases and ted wolffish (Anarhichas minor), turbot (Scoph- development of skin ulcers or lesions on the thalmus maximus), wrasse (Labrus berggylta), sides of the body. The gills are often pale with European eel (Anguilla anguilla ) and gold- petechial haemorrhages. Internal features like fish (Carrassius auratus). Furthermore, atypical hyperaemia of serosal surfaces, haemorrhages A. salmonicida has been detected in ulcerated in internal organs and mucosa are frequently wild fish of various marine and fresh water detected. The course of the disease can be species. These include species such as sand- peracute, acute, subacute or chronic as de- eels (Ammodytes lancea and Hyperoplus lanc- scribed for classical furunculosis. Pure cul- eolatus), flounder (Platichthys flesus), dab tures of the bacterium can be obtained from (Limanda limanda), plaice (Pleuronectes plat- internal organs, for example the kidney, spleen essa), cod (Gadus morhua), haddock (Melano- and heart (Kimura, 1970; Paterson et al., 1980; grammus aeglefinus), whiting, (Merlangius Morrison et al., 1984; Groman et al., 1992; Olivier, 1992; Helleberg et al., 1996; Gud- ther possess serine gelatinase nor a haemo- mundsdóttir et al., 1997; Helgason et al., lytic activity. On the other hand the ECP of the ssp. salmonicida strain contained a 70 kDa In many cases, however, infections by atypi- serine protease, P1, and a glycerophospholipid: cal A. salmonicida (e.g. in salmonids, cyprinids, cholesterol acyltransferase, GCAT, which are eels, wild flatfish species, etc.) cause an ulce- the major exotoxins of typical A. salmonicida rative disease with more superficial variety of strains, but a metallo-caseinase was not de- pathological changes than furunculosis, start- tected in its ECP. The main difference of the ing as small haemorrhages in skin and pro- gross pathology detected was that skin lesions gressing to multiple skin lesions. Other bacte- caused by the ssp. achromogenes strains were rial species often become involved, as sec- usually shallower with haemorrhages at the ondary infections of the ulcers often occur, edges, whereas lesions induced by ssp. sal- and the cause of death is not always clear. It monicida extended deeper into the muscle and has been observed that the fish can die al- haemorrhages were more extensive. Histopatho- though a bacteremia is not detected. In ad- logical changes, in both cases, were similar to vanced stages of infection, however, bacteria those typical for acute furunculosis, showing are also found in the blood and internal or- bacterial colonization in a variety of sites (skin, gans. The ulcers can be located anywhere on gills, spleen, pancreas, kidney, heart, brain and the body surface, although they are most fre- liver) with localized cellular necrosis. How- quently found on the flanks. Infected fish may ever, tissue damage induced by ssp. salmoni- show inappetance, lethargy, loss of orienta- cida was usually more severe and with more tion, and abnormal swimming behavior. Bac- extensive necrosis (Gudmundsdóttir et al., terial isolation should be done from recently developed skin ulcerations as well as from in- ternal organs (Mawdesley-Thomas, 1969; Fijan, developed host reaction to atypical A. salmon- 1972; Bootsma et al., 1977; Nakai et al., 1989; icida including a marked leucocyte response Groman et al., 1992; Austin and Austin, 1993; with resultant encystment of the bacteria. His- topathological changes are uniform in the vari-ous tissues and characterized by granuloma Comparison of A. salmonicida infections in formation. Centrally in the granuloma are colo- nies of bacteria usually with necrosis encir- The variation in extracellular virulence fac- cled by many layers of epitheloid cells, sur- tors produced by atypical and typical A. salmon- rounded with a thin layer of fibroblasts. These icida, and within the heterogeneous group of changes are very different from those caused atypical strains, may explain the different pa- by atypical A. salmonicida strains in salmonids, thology caused by various A. salmonicida even when the isolated strains are akin as re- strains. Another reason is that the various hosts gards biochemical characters and exotoxin pro- duction (Helgason et al., 1997).
in wild Atlantic salmons (2–3 kg) from an Ice- landic river, that were naturally infected with Although A. salmonicida has been known as a typical or atypical A. salmonicida, respective- fish pathogen and studied for over 100 years, ly. The isolated organisms were classified as its virulence mechanisms are still only partly A. salmonicida ssp. achromogenes and sal- understood. The application of sophisticated monicida, respectively. A 20 kDa metallo-ca- biochemical techniques in the recent years, seinase, AsaP1, was detected in the ECP of however, continues to yield considerable new the ssp. achromogenes strain, but it did nei- information. In a systemic infection like fu- runculosis and related diseases, the successful nants that constitute the serological specificity.
pathogen must have properties that allow it to The characteristic endotoxic effects of Gram- avoid, withstand, or overcome the non-spe- negative bacteria are associated with the lipid cific and immunospecific defense mechanisms of the host. Atypical and typical strains of A. tremely resistant to the lethal effects of LPS salmonicida have been reported to share cell- (Berczi et al., 1966). The LPS of A. salmon- associated antigens like lipopolysaccharide icida have a structural function in assembly (LPS) and outer membrane proteins, but ex- and maintenance of the A-layer (Noonan and tracellular virulence factors produced by typi- Trust, 1995b) and are found to be species spe- cal and atypical strains are more different (Aus- The A-layer protein and LPS of typical and atypical strains are serologically cross reac- tive and the most potent A. salmonicida anti- The cell envelope of Gram-negative bacteria gens in inducing antibody response in Atlan- consists of a cell membrane, a peptidoglycan tic salmon (Bjørnsdóttir et al., 1992), although layer and an outer membrane. A. salmonicida anti A-protein antibodies have not been shown has a surface protein layer (A-layer) external to be protective (Gudmundsdóttir and Magna- to the outer membrane which is interspersed between the repeated o-polysaccharide (O-anti-gen) subunits of the LPS (Udey and Fryer, 1978; Evenberg et al., 1985). The A-layer plays One mechanism involved in host defense in- an important role in the virulence of the or- volves the binding of free iron to proteins such ganism as mutants lacking it have lost their as transferrin to create iron-restricted condi- tions within the host. As iron is essential for 1995a). Like many other bacterial surface layers bacterial growth, a successful pathogen must the A-layer is composed of a single protein have mechanisms enabling it to compete with subunit, the A-protein (or VapA), which as- the iron binding proteins in the serum and ex- sembles on the surface to form a tetragonal tracellular fluids of the host. Such mechanisms array surrounding the entire cell (Udey and include the production of siderophores and Fryer, 1978). The structural gene for the A- the induction of outer membrane proteins ca- layer protein has been cloned (Chu et al., 1991).
pable of binding iron containing host proteins.
The A-layer is multifunctional. It serves to Typical and atypical strains of A. salmonicida protect A. salmonicida from serum effects possess an effective high-affinity iron-uptake (Munn et al., 1982), the action of proteases mechanisms. However, atypical and typical (Chu et al., 1991) and from phagocytosis (Trust A. salmonicida strains use some different mech- et al., 1996). Furthermore, it facilitates bind- anisms to acquire iron under conditions of iron- ing to certain porphyrins, immunoglobulins and restriction. Hirst and Ellis (1994) identified a range of extracellular matrix proteins (Trust four iron regulated outer membrane proteins et al., 1996). Results by Noonan and Trust (IROMPs) that are shared by typical and atypi- (1995b) indicate that the primary requirement cal strains. Furthermore, their results indicated for the A-layer of A. salmonicida may be at that the IROMPs of A. salmonicida are impor- the early stages of infection. The LPS endo- tant for induction of antibodies that are bac- toxin is an essential component of the outer tericidal for a virulent A. salmonicida strain membrane. It is composed of three moieties: in vitro and protective against furunculosis.
lipid A, a core oligosaccharide and the O-an- Only typical A. salmonicida strains produce tigen, which is exposed at the cell surface.
siderophore for iron uptake, but the atypical The O-antigen carries the antigenic determi- strains that have been tested so far, acquire iron in the host by a siderophore-independent tease was detected in the reference cultures for A. salmonicida ssp. achromogenes and 24fresh isolates from 10 species of fish in the Nordic countries, Scotland and Canada. A to- It is well known that environmental conditions tal of 19 strains did not produce detectable affect synthesis of certain bacterial compo- amounts of either of the proteolytic exotoxins nents, a fact that must be kept in mind when (Austin et al., 1998). A lethal toxin that is results from different laboratories are com- neither caseinolytic nor haemolytic and sev- pared. Studies of the virulence properties of eral other proteases, with unknown pathogenic A. salmonicida have mainly been performed activity, have also been detected in the ECPs using ssp. salmonicida strains grown in vitro of atypical A. salmonicida strains (Gudmunds- and samonids as the host. Most of the avail- able information has been obtained by biochem- The exact roles played by the various viru- ical characterization of secreted enzymes and lence factors of A. salmonicida identified to toxins, but recently analysis at the genetic level date is unclear. When injected into fish, puri- fied factors or combinations of factors can re- Extracellular products (ECP) of some atypi- sult in disease signs similar to those observed cal strains have been reported to be lethal for during infection with A. salmonicida. How- salmon and carp. The toxicity of the ECPs is ever, more research is needed before func- of a proteinous nature and some virulence re- tions can be assigned to each of the virulence lated factors have been identified (Gudmunds- dóttir, 1997). It has been shown by genetic methods, that there are atypical strains that produce the P1 protease and/or the GCAT cy-totoxin, which are the major exotoxins of Atypical A. salmonicida strains carry plasmids typical strains, but some strains do not pro- which are different from plasmids of typical duce either of these enzymes (Austin et al., strains. A great variability has been observed 1998). A 20 kDa metallo-caseinase, AsaP1, in plasmid content of different atypical strains has been isolated from the ECPs of some atypical (Austin et al., 1998). None of the identified strains and identified as the major exotoxin of virulence factors of A. salmonicida have been a group of atypical strains including type strains linked to a specific plasmid (Noonan and Trust, for A. salmonicida ssp. achromogenes (Gud- mundsdóttir and Magnadóttir, 1997; Gunn- laugsdóttir and Gudmundsdóttir, 1997). TheAsaP1 toxin is a powerful mitogen of Atlantic Atypical A. salmonicida strains have occasion- salmon leukocytes but does not exert cyto- ally been isolated from fish without any dis- toxic activity (Gudmundsdóttir, et al., 1995).
ease signs as well as diseased fish from na- It has been shown that AsaP1 can induce pro- ture, besides being associated with epizoot- tective immunity against atypical furunculo- ics in wild fish populations. Losses in farmed sis in Atlantic salmon (Gudmundsdóttir and salmonids and non-salmonids are increasingly Magnadóttir, 1997). In a study where 52 fresh associated with atypical A. salmonicida infec- isolates of atypical A. salmonicida were in- tions, presumably because of the intensive fish vestigated, the P1 protease was detected in farming together with an increase in diagnos- the reference cultures of subspecies salmon- tic awareness and capability (Wiklund and icida and smithia and 10 of the fresh iso- Dalsgaard, 1998). As with infections caused lates, originating from 6 different fish species by typical A. salmonicida clinical disease out- and 6 geographical locations. The AsaP1 pro- breaks due to atypical strains usually occur following stress-inducing events, like handling viable bacteria can also be spread by aerosols in the hatchery, overpopulation, rapid tempera- (Wooster and Bowser, 1996). Ribotyping is ture or water flow changes, or following transfer suggested as a valid method to study the epi- of captured fish to cages. The explanation is demiology of infections caused by atypical A. presumably that covertly infected fish gets sick salmonicida (Pedersen et al., 1996).
The source of the infection usually remains uncertain. It is known that the organism can Diagnosis of A. salmonicida infection is based be transmitted horizontally both between and upon clinical signs of disease and isolation within fish populations, which includes con- and identification of the causative agent. The tact with contaminated water and infected fish diagnosis of atypical A. salmonicida infections in addition to possible infection via the gastro- is more difficult than the diagnosis of typical intestinal tract (McCarthy, 1980). Transmis- furunculosis as clinical and pathological signs sion by diseased fish escaping from infected vary and some of the strains are fastidious and farm stocks held in both fresh- and seawater slow growing on initial isolation. As many bac- are a source of infection and in many cases terial infections in fish can manifest with similar infections have been traced to transport of fish.
symptoms, the isolation and identification of The transport of live fish, like ornamental fish and baithfish for angling, and fish eggs around the world has disseminated atypical strains and additional possibility of successful isolation of the bacterium. In addition to the anterior Australia (Whittington et al., 1987) and Chile part of the kidney samples can be taken from (Sandra Bravo, personal communication) by liver, heart, spleen, intestine, gills, skin mu- cus and ulcers. For fish exhibiting skin le- Compared to typical A. salmonicida there sions, material from the periphery of the le- is limited information available on transmis- sions should be inoculated into the bacterio- sion and survival of atypical strains in water.
logical medium (Bernoth, 1997). Often there The available data indicate that the bacteria are contamination problems on agar plates in- survive better in brackish and seawater than in cubated with samples taken from the external freshwater. The data must, however, be inter- organs and the intestine. It has, however, been preted with caution, as they are based only on stated that samples from skin-and gill mucus laboratory results, and survival of various atypi- may be more reliable than those from internal cal strains in natural habitats may be distinct organs and the advantage is also that samples (Wiklund, 1995). In Norway, Sweden and Fin- can be taken without killing the fish (Benedikts- land outbreaks of atypical furunculosis have dóttir and Helgason, 1990; Bernoth, 1997).
occurred both in freshwater and seawater. In In order to detect covert infections in fish Iceland outbreaks of atypical furunculosis in sampling from gills and intestine is recom- farmed salmonids almost always occur in brack- mended. Stress-testing is required to improve ish environment. Furthermore, experimental the sensitivity of detection. The fish is then infection of salmon by cohabitation with fish stressed by an injection of corticosteroid (0.25 infected by an Icelandic isolate has been es- mg/kg prednisolon acetat) and kept for 14 days tablished in brackish but not in freshwater in 18°C before the bacterial examination is (Gudmundsdóttir and Gudmundsdóttir, 1997).
carried out (Bernoth, 1997; Hiney, 1997).
Although fish are still regarded as the main For the primary isolation of the bacterium vector in transmission of A. salmonicida, para- cultivation on blood agar (BA), tryptic soy sites and marine plankton may also be involved agar (TSA) or brain heart infusion agar (BHIA) (Enger, 1997) and recent results indicate that oil emulsion. Currently all commercially avail- mented with coomassie brilliant blue (CBB) able A. salmonicida vaccines are produced from typical A. salmonicida strains for prevention protein stain, which is absorbed by the A-layer- of classical furunculosis in salmonids. There protein and gives A. salmonicida colonies a is, however, evidence of cross protection against blue color after 2–7 days incubation. For the atypical furunculosis in Atlantic salmon vac- isolation of fastidious strains prolonged incu- cinated with commercial oil based furunculo- bation (7–14 days) at 20–22°C is required.
sis vaccine (Jones et al., 1996; Gudmunds- Morphological and biochemical characteris- dóttir and Gudmundsdóttir, 1997). As diseases tics, as described above for atypical A. sal- caused by atypical strains are of emerging im- monicida, are used to identify the bacterium.
portance worldwide, the prospects of their con- When the amount of bacteria in fish tissue is trol by vaccination need to be considered. Ice- very low more sensitive methods, such as im- landic fish farmers have since 1992 success- munofluorescent antibody technique (IFAT en- fully used an autogenous A. salmonicida ssp.
achromogenes bacterin to vaccinate salmonids can be used (Bernoth, 1997; Wiklund and Dals- by injection and recently halibut farmers in Norway have started to use an autogenous in-jection vaccine against atypical furunculo- The etiology of bacterial diseases is diverse.
The presence of the relevant pathogen, a sus- ceptible host and a conductive environment Chemotherapy has been the most used method are the prerequisite for the development of a for treatment of atypical A. salmonicida in- particular disease. Therefore, husbandry fac- fections. The most commonly used antimicro- tors like hygiene, disinfection of transported bial agents are oxolinic acid, oxytetracycline material, nutrition, water quality, the stocking and trimethoprim-sulfamethoxazole. Other anti- density, handling and disturbance are factors microbial agents that have been shown to be that are highly important for the fish health.
effective against these pathogens are i.e. chlor- The environment can affect the fish defense systems through a variety of mechanisms. Stress ciprofloxacin (Wiklund and Dalsgaard, 1998; is known to impair the immune response sig- Heo and Seo, 1996). Atypical A. salmonicida nificantly. The stress response in teleost fish strains are frequently resistant to amoxycillin, is induced by environmental factors although which in some places is used to treat classical factors inherent to the fish itself, like matu- furunculosis (Barnes et al., 1991). Atypical rity, are also involved. Furunculosis and re- strains with resistance to antibiotics have lated diseases are one of the best examples of emerged following extensive use of chemo- diseases strongly influenced by environmen- therapeutics in the aquaculture and it has been tal factors. Good husbandry is therefore of shown that a plasmid encoding for multiple great importance for preventing outbreaks of resistance can be transferred from atypical A. salmonicida to some marine bacteria (Sandaa measure against furunculosis of salmonids. Al- though, the development of furunculosis vac- have sometimes been used to control infec- cines has been one of the great challenges to tions by atypical strains, especially at the first researchers for many years, the first effective outbreak of the disease on a farm where the ones were not on the market until after 1992.
risk for reinfection from feral fish is consid- These are injectable bacterins adjuvanted with ered to be low (Wichard et al., 1989).
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