clonality of vibrio anguillarum strains isolated from fish from the scandinavian countries, sweden,...

Journal of Applied Microbiology 1999, 86, 337–347

Clonality of Vibrio anguillarum strains isolated from fish fromthe Scandinavian countries, Sweden, Finland and Denmark

K. Pedersen 1*, I. Kuhn2, J. Seppa nen3, A. Hellstro¨ m4, T. Tiainen 1, E. Rimaila-Pa rnanen3

and J.L. Larsen 1

1Laboratory of Fish Diseases, The Royal Veterinary and Agricultural University, Frederiksberg, Denmark,2Microbiology and Tumorbiology Center, Karolinska Institute, Stockholm, Sweden, 3National Veterinary andFood Research Institute, Helsinki, Finland, and 4National Veterinary Institute, Uppsala, Sweden

6839/08/98: received 10 August 1998, revised 11 September 1998 and accepted 21 September 1998

K. PEDERSEN, I. KUHN, J. SEPPANEN, A. HELLSTROM, T. TIAINEN, E. RIMAILA-PARNANEN

AND J.L. LARSEN. 1999. In order to investigate whether outbreaks of vibriosis in the Balticregion were caused by the spread of certain pathogenic clones, 291 Vibrio anguillarumisolates from Finland (n � 156), Sweden (n � 88) and Denmark (n � 47) werestudied with respect to serogroup, ribotype, plasmid content, and biochemicalphenotypes as expressed with the PhenePlate (PhP) typing system. For comparison, 54 V.anguillarum serogroup O1 from other countries worldwide were included. Most isolatesfrom Finland, Sweden and Denmark belonged to serogroup O1 (255), followed by O2 (30).Four Finnish isolates cross-reacted strongly with antisera against two new serogroupsVaNT2 and VaNT4, whereas two strains were non-typeable. The serogroup O1 isolatesdisplayed ten different ribotype patterns, whereas the other strains were considerablymore diverse with respect to ribotypes. Most of the O1 isolates carried the 67 kbvirulence plasmid and a group of Finnish isolates, in addition, carried an 86 kb plasmid.Additional plasmids with molecular weights of 63, 76, 135 or 260–290 kb were found insingle O1 isolates. With few exceptions, strains of serogroup O2 either had no plasmidsor carried one or two small plasmids. PhenePlate typing revealed considerable diversitywithin the species, serogroup O1 being the most homogeneous. A few PhP types weredominant, whereas other types were observed only in one to four isolates. The prevalence ofthe different types changed significantly from one year to another but in Finland, oneclonal lineage became increasingly important from 1992 (20% of isolates) to 1996(80%). Remaining clones were mostly restricted to specific geographic areas. By clusteranalysis, it was demonstrated that most of the isolates from Finland, Sweden and Denmarkbelonged to two clusters, and most of the strains from Southern Europe fell into twoother, distinct clusters. Most isolates from the UK, North America, Chile andTasmania grouped together in a distinct cluster. For the typing of V. anguillarum, O-serotyping should be the primary method. For isolates belonging to serogroupsother than O1, plasmid profiling in combination with ribotyping gives a very gooddiscrimination between strains, whereas for serogroup O1, another method is required. It isconcluded that PhP typing is a tool that provides a good discrimination between O1isolates.

Correspondence to: Karl Pedersen, Laboratory of Fish Diseases, Department ofVeterinary Microbiology, The Royal Veterinary and Agricultural University,Bulowsvej 13, DK-1870, Frederiksberg C, Denmark (e-mail: [email protected]).*Present address: Danish Veterinary Laboratory, Hango�vej 2, DK-8200 AarhusN, Denmark.

© 1999 The Society for Applied Microbiology

INTRODUCTION

Vibrio anguillarum is an important fish pathogen in manycountries where fish are grown in aquaculture (Austin andAustin 1993), including the coastline of the Baltic Sea inFinland, Sweden and Denmark. Vibrio anguillarum is still

338 K. PEDERSEN ET AL.

causing outbreaks of disease in Finnish and Swedish farmedsalmonids in spite of vaccination programmes. In Finland,vibriosis is enzootic in all brackish waters except for thenorthern-most part of the Gulf of Bothnia, where the salinityis close to 0%. Vibrio anguillarum is isolated every year fromdiseased farmed fish in the Gulf of Bothnia, the Baltic Seaand the Gulf of Finland. In Sweden and Denmark, vibriosishas previously been a problem on fish farms, but the fre-quency of outbreaks has decreased during the 1990s, probablydue to the implementation of proper prophylactic measures,including vaccination. Vibrio anguillarum is also a naturallyoccurring bacterium in the marine environment and can beisolated in large numbers during the summer season fromwater, sediment and plankton (Larsen 1985, 1990) as well asfrom the intestine, gills and mucus of healthy fish (Larsen1990 ; Pedersen and Garcia 1996). However, these environ-mental isolates are usually not virulent for fish (Larsen 1990 ;Pedersen and Larsen 1995 ; Pedersen and Garcia 1996 ;Pedersen et al. 1997). An environmental habitat for the viru-lent forms, and the way in which they are spread from onefish farm to another, is not known in any detail. The purposeof the present study was to evaluate diversity among V.anguillarum strains isolated from a certain geographical regionover time, and to follow the clonality of V. anguillarum in thisarea. For this purpose, 291 V. anguillarum isolates from fishalong the coast of the Baltic Sea in Sweden, Finland andDenmark were studied. For comparison, 54 strains fromother regions were included.

MATERIALS AND METHODS

Bacterial strains and culture conditions

A total of 156 Finnish, 88 Swedish and 47 Danish strains ofV. anguillarum, isolated in the Baltic area during the period1982–1995, was investigated. All strains, except for five, werefrom diseased fish and most of them from outbreaks of vib-riosis in fish farms. Only one isolate from each outbreakwas included. For comparison, 54 strains of V. anguillarumserogroup O1 from other countries, all isolated from diseasedfish, were included : Norway (n� 5), Italy (n� 28), Greece(n� 3), Spain (n� 3), France (n� 3), Germany (n� 1),UK (n� 2), Canada (n� 4), USA (n� 2), Chile (n� 1),Tasmania (n� 1) and Taiwan (n� 1). Therefore, the totalnumber of isolates included in the study was 345.

Serotyping

O-serotyping on the basis of heat stable O-antigens was car-ried out as described by Larsen et al. (1994) using rabbitantisera against the serogroups O1–O10 (So�rensen andLarsen 1986) and the four additional serogroups, VaNT2,

© 1999 The Society for Applied Microbiology, Journal of Applied Microbiology 86, 337–347

VaNT4, VaNT5 and VaNT7, introduced by Austin et al.(1995).

Ribotyping

rRNA gene restriction pattern analysis (ribotyping) was per-formed as described by Pedersen and Larsen (1993) andPedersen et al. (1996). Isolated total DNA was digested withHindIII and subjected to electrophoresis in 0·8% agarose gelsin TAE (Tris 40 mmol l−1, sodium acetate 5 mmol l−1, EDTA1 mmol l−1) buffer, pH 7·8. Separated DNA fragments wereblotted onto nylon membranes (Hybond-N, Amersham, UK,or Magna Nylon, Micron Separations Inc., Westborough,MA, USA) and hybridized with a digoxigenin-labelled probecomplementary to 16S and 23S RNA from Escherichia coli.Labelled fragments were visualized by incubation with alka-line phosphatase-labelled anti-digoxigenin immunoglobulin(Boehringer Mannheim) followed by the addition of nitrobluetetrazolium and 5-bromo-4-chloro-3-indolyl-phosphate(Boehringer Mannheim). The ribotype patterns of serogroupO1 strains were given numbers referring to the patternspreviously described by Pedersen and Larsen (1993), whereasthe serogroup O2 strains were given arbitrary pattern des-ignation letters A–R. The remaining strains, not belongingto serogroup O1 or O2, were given arbitrary pattern des-ignations, l, m, n, p, s and v.

Plasmid profiling

Plasmids were isolated by the method of Kado and Liu (1981)and separated by electrophoresis in 0·8% agarose gels inTAE buffer. DNA was visualized by staining with ethidiumbromide and photographed at 254 nm u.v. transillumination ;plasmid molecular weights were then calculated as describedby Tiainen et al. (1995). Distinction between covalentlyclosed circular forms and open circular forms was done bythe u.v. irradiation method described by Hintermann et al.(1981).

PhenePlate (PhP) typing

Biochemical fingerprinting using the PhenePlate system(BioSys inova, Stockholm, Sweden) was carried out asdescribed by Kuhn et al. (1990, 1996, 1997) and Mollby etal. (1993). Briefly, the quantitative kinetics of 11 biochemicalreactions, selected to give high discriminatory power for V.anguillarum strains, were measured spectrophotometrically at620 nm in microtitre plates. The 11 biochemical reactionswere : degradation of L-arabinose, galactose, mannose, cel-lobiose, trehalose, sucrose, gentobiose, sorbitol, mannitol,gluconate and starch. Plates were incubated at 30 °C and thereactions read after 16, 40 and 64 h. The mean absorbance ineach well was calculated and the similarity between the iso-

CLONALITY OF VIBRIO ANGUILLARUM 339

Table 1 Distribution of ribotypes, plasmids and PhP types onserogroup O1 Vibrio anguillarum isolates from Sweden, Finland andDenmark. The number of strains is shown in brackets—–––––––––––––––––––––––––––––––––––––––––––––––––––––

Country Ribotype Plasmids PhP type—–––––––––––––––––––––––––––––––––––––––––––––––––––––Finland (151) 1 (111) pJM1 (107) 1 (79)

2b* (1)3 (6)4 (20)Single† (1)

73 kb (1) 1 (1)– (3) 1 (3)

4 (40) rjM1 (25) 1 (3)2 (1)4 (13)6 (7)S4/S71 (1)

pJM1, 86 kb (13) 2 (10)2b (2)4 (1)

45 kb, pJM1 (1) 4 (1)– (1) 4 (1)

Sweden 1 (29) pJM1 (28) 1 (20)(n � 73)

1A (1)2 (1)4 (5)4B (1)

pJM1, 135 kb (1) 1A (1)4 (33) pJM1 (32) 1 (1)

4 (13)4B (7)4C (1)D4 (2)D28 (2)D82 (2)D99 (2)Single (2)

63, 76 kb (1) 4 (1)6 (11) pJM1 (10) 1 (9)

NF (1)260–290 kb (1) 1 (1)

—–––––––––––––––––––––––––––––––––––––––––––––––––––––

lates was then calculated as the correlation coefficient betweenthe 11 reactions. Strains were assigned to the same PhP typeif they had a similarity higher than 0·975 to each other.Clustering of strains was performed by the unweighted pairgroup method with arithmetic averages (UPGMA) (Sneathand Sokal 1973). Phenotypic diversity was calculated as theSimpson’s index of diversity (Di) (Atlas 1984). PhenePlatetypes were given specific designations if more than one strain


Table 1 — continued—–––––––––––––––––––––––––––––––––––––––––––––––––––––

Country Ribotype Plasmids PhP type—–––––––––––––––––––––––––––––––––––––––––––––––––––––Denmark 1 (24) pJM1 (20) 1 (16)(n � 31)

3 (3)4 (1)

pJM1, 90 kb (2) 1 (1)Single (1)

90 kb (1) 1 (1)– (1) 1 (1)

2 (1) 4·5 kb (1) Single (1)6 (1) 54 kb, pJM1 (1) 1 (1)7‡ (1) – (1) Single (1)

10‡ (3) – (3) 13 (3)11‡ (1) – (1) 13 (1)

—–––––––––––––––––––––––––––––––––––––––––––––––––––––

* 2b means a PhP type similar to PhP type 2, but not quiteidentical.† Single means that this strain has a unique PhP type not sharedwith any other strain.‡ These strains are environmental non-pathogenic strainsisolated from water (n � 1), or from mucus (n � 2), faeces (n � 1)or gills (n � 1) of healthy rainbow trout.

had this type ; strains with unique PhP types were designatedas single (S).

RESULTS

The distribution of the isolates from Sweden, Finland andDenmark on serotypes, ribotypes, plasmid content and PhPtypes is shown in Table 1 (serogroup O1 only) and Table 2(other serogroups), and of the strains from other countries,in Table 3.

The Finnish strains were all isolated from diseased sal-monid fish, mostly rainbow trout, during the 5 year period1992–1996. The majority of these isolates belonged to sero-group O1 (151 of 156), whereas only one strain was O2. Fourisolates cross-reacted strongly with antisera against the newserogroups VaNT2 and VaNT4. The serogroup O1 isolatesbelonged to the two ribotype patterns 1 (111 of 151) and 4(40 of 151). Irrespective of ribotype, all but five of theseisolates carried the 67 kb pJM1 plasmid. One isolate carrieda 73 kb plasmid, although this was shown by restrictionanalysis to be a derivative of pJM1, and four serogroup O1strains had no plasmids. Thirteen isolates carried an 86 kbplasmid in addition to the 67 kb plasmid. These 13 isolatesall belonged to ribotype pattern 4. A total of eight PhP typeswas recorded among the serogroup O1 isolates, of which PhPtype 1 (n� 86), 2 (n� 11), 4 (n� 36) and 6 (n� 7) werethe most frequent. Among the strains carrying the 86 kb


Table 2 Distribution of serotypes, ribotypes, plasmids and PhPtypes on Vibrio anguillarum isolates from Sweden, Finland andDenmark not belonging to serogroup O1. The number of strainsis shown in brackets—–––––––––––––––––––––––––––––––––––––––––––––––––––––

Country Serotype Ribotype Plasmids PhP type—–––––––––––––––––––––––––––––––––––––––––––––––––––––Finland O2 B – D65(n � 5) VaNT2/VaNT4n 6·5 kb 13

VaNT2/VaNT4p – SVaNT2/VaNT4s 2·6 13VaNT2/VaNT4v – S

Sweden O2 A – D55(n � 15) O2 B 7·8 kb D65

O2 B – D65O2 C – 7O2 C – SO2 C 5·5 kb 7O2 D 22 kb SO2 D – SO2 E 4·2 kb SO2 E 4·8 kb 7O2 F – D55O2 G – SO2 H 4·2 kb 7NT l – D55NT m – S

Denmark O2 I – D32(n � 16) O2 I – S

O2 I – D32O2 I – SO2 J 4·2 SO2 J 4·2, 5·1 D40O2 J 4·2 D40O2 K 4·2 D36O2 K 4·2 D36O2 L 5·1 1O2 M – SO2 N ×200 SO2 O – D43O2 P – D43O2 Q – 13O2 R – S

—–––––––––––––––––––––––––––––––––––––––––––––––––––––

plasmid, 12 out of 13 belonged to PhP type 2 or 2b. Amongribotype 1, one PhP type (PhP type 1) was dominant (83 of111) and a second type (PhP type 4) was found in 20 of 111isolates. Among ribotype 4, PhP type 4 was the most common(16 of 40) but also, PhP type 2 was found in 13 isolates. Ittherefore seems that although some correlation existedbetween ribotype and PhP type, these two properties did notalways coincide.


Table 3 Distribution of ribotypes, plasmids and PhP types onVibrio anguillarum serogroup O1 isolates from outside theBaltic region. The number of strains is shown in brackets—–––––––––––––––––––––––––––––––––––––––––––––––––––––

Country Ribotype Plasmids PhP type—–––––––––––––––––––––––––––––––––––––––––––––––––––––Norway (5) 1 (4) pJM1 (4) 1 (2)

6 (1)Single (1)

6 (1) 33 kb, pJM1 (1) 1 (1)Italy (28) 1 (24) pJM1 (19) IT3 (19)

89, 90 kb (1) IT4 (1)80, 90 kb (1) IT4 (1)pJM1, 86 kb (3) IT4 (2)

Single (1)3 (4) pJM1 (4) IT5 (4)

Greece (3) 1 (3) pJM1 (3) IT3 (2)Single (1)

Spain (3) 1 (3) pJM1 (3) IT5 (3)France (3) 1 (2) pJM1 (1) Single (1)

36 kb, pJM1 (1) IT3 (1)5 (1) pJM1 (1) IT5 (1)

Germany (1) 1 (1) pJM1 (1) 1 (1)UK (2) 1 (2) pJM1 (1) Single (1)

5·3, 50 kb (1) Single (1)Canada (4) 4 (2) pJM1 (2) Cn7 (2)

13 (2) pJM1 (2) Cn6 (2)USA (2) 1 (2) pJM1 (1) 1 (1)

50, pJM1 (1) Single (1)Chile (1) 1 (1) pJM1 (1) NF (1)Tasmania (1) 1 (1) pJM1 (1) Single (1)Taiwan (1) 1 (1) pJM1 (1) IT4 (1)—–––––––––––––––––––––––––––––––––––––––––––––––––––––

Only five of the Finnish strains were of single PhP types,including the single serogroup O2 isolate, but this strainshared PhP type as well as ribotype with two Swedish strains.

The four VaNT2/VaNT4 cross-reacting isolates all haddifferent, although very similar, ribotype patterns. Two ofthem shared PhP type whereas the other two had unique PhPtypes (Table 2). These four strains were isolated on fourdifferent fish farms.

The Swedish strains were mostly isolated from diseasedrainbow trout but 16 were from other fish species. Like theFinnish isolates, the majority belonged to serogroup O1 (73of 88). Thirteen isolates belonged to serogroup O2, whereastwo isolates were non-typeable. The serogroup O1 strainsbelonged to three different ribotypes, 1 and 4 as in Finland,and ribotype 6, and they all carried plasmids. Out of 73serogroup O1 isolates, 71 had the pJM1 plasmid. One ofthese isolates had an additional large plasmid of 135 kb.Furthermore, one isolate had a very large plasmid of 260–290 kb. The last O1 isolate had two plasmids, 63 and 76 kb,one of which was a derivative of the 67 kb pJM1 plasmid. The


Swedish O1 isolates displayed more diversity with respect toPhP types than the Finnish isolates, but the most commontypes were the same as the most common Finnish PhP types.The major PhP types were 1 and 4 containing 31 and 19isolates, respectively. Only eight isolates were single typesand five of those were O2 strains. The 13 serogroup O2isolates were divided into eight different ribotypes, whichsometimes corresponded to the PhP types and sometimes not(Table 2). One of these ribotypes, B, was identical to theribotype of the single Finnish O2 isolate, and the three Swed-ish and Finnish isolates belonging to this ribotype also hadidentical PhP type.

The Danish strains belonged to serogroup O1 (31 of 47)and O2 (16 of 47). Most of the strains (39 of 47) were isolatedfrom rainbow trout, but one strain was isolated from eel andtwo from turbot. Five strains were environmental isolates.Among the O1 strains, six different ribotypes were found(Table 1). However, the strains with the ribotypes 7, 10and 11 were the environmental strains, and the strain withribotype pattern 2 has later been shown to be non-pathogenicto fish, although it was originally isolated from the kidney of arainbow trout (Pedersen et al. 1997). Thus, all fish pathogenicstrains belonged to the two ribotypes 1 and 6. Most of theserogroup O1 isolates carried the pJM1 plasmid, and most ofthese isolates shared PhP types with isolates from Finlandand Sweden. The 16 O2 strains belonged to 10 differentribotypes, none of which were found in Swedish or Finnishisolates (Table 2).

Twelve of the total number of 30 O2 strains carried one ortwo small plasmids of 4·2–7·8 kb, one carried a medium sizedplasmid of 22 kb, and one strain a large plasmid ×200 kb.

Ribotype 6 was found only in strains from Sweden(n� 11), Norway (n� 1) and Denmark (n� 1). Strains ofthis ribotype displayed interesting PhP reactions by showingslightly varying results at repeated testings. However, theirPhP types were most similar to PhP type 1 and they weretherefore allocated to this type. Such variations were neverrecognized in their ribotype or plasmid profiles.

Although many of the PhP types among the O1 isolateswere found in all three countries, some were found onlyin one country and there was considerable variation in theprevalence of these PhP types between countries and betweenyears of isolation. The year of isolation was not known for allDanish isolates, but Figs 1 and 2 show the prevalence of themost important combinations of ribotypes and PhP typesin Finland and Sweden, respectively, in different years. InFinland, the clonal lineage of strains with ribotype 1 and PhPtype 1 has become considerably more prevalent during theperiod from 1992 to 1996, increasing from approximately30% of the isolates to approximately 80%. During the sameperiod, the proportions of the other clonal lineages havedecreased accordingly. The combination of ribotype 4 andPhP type 2, which are the strains carrying pJM1 and the


70

1992

Perc

en

tag

e o

f is

ola

tes

Time period

50

1993 1994 1995

60

40

30

20

10

1996

Fig. 1 Prevalence of combinations of the most importantribotypes and PhP types in Finland during the period 1992–1996.P and R indicate PhP type and ribotype, respectively : ( ), P1-R1 ; ( ), P4-R1 ; ( ), P4-R4 ; (Ž), P2-R4p ; (�), other

60

85–86

Perc

en

tag

e o

f is

ola

tes

Time period

50

87–90 91–92

40

30

20

10

93–95

Fig. 2 Prevalence of combinations of the most importantribotypes and PhP types in Sweden during the period 1985–1995.P and R indicate PhP type and ribotype, respectively : ( ), P1-R1 ; ( ), P4-R1 ; ( ), P4-R4 ; (Ž), P1-R6 ; (�), other O1

86 kb plasmid, were found only in 1993, 1994 and 1996(Fig. 1). Considerable variations in prevalence were alsonoticed among the Swedish serogroup O1 isolates. The com-bination of ribotype 1 and PhP type 1 first decreased fromalmost 40% of the V. anguillarum strains isolated in 1985–1986 to 0 in 1991–1992, and then increased again to 40% in1993–1995 (Fig. 2).

The serogroup O1 strains from outside Finland, Swedenand Denmark displayed marked differences from the O1strains from these three countries with respect to PhP typesand sometimes also ribotypes (Table 3). Only four of the fiveNorwegian isolates, one isolate from the USA and one isolatefrom Germany shared combinations of ribotype and PhP typewith strains from Finland, Sweden and Denmark. Remainingisolates had different PhP types. Most isolates belonged toribotype 1, but ribotypes that were not detected among thestrains from Finland, Sweden and Denmark were found instrains from Italy (ribotype 3), France (ribotype 5) and Can-ada (ribotype 13, which has not previously been described).


Most of the strains from Italy, Greece, Spain and France (34of 37) belonged to three PhP types, IT3, IT4 and IT5. Apartfrom a single isolate from Taiwan, which had PhP type IT4,these PhP types were not found outside the Mediterraneanarea in the present material. The Canadian isolates belongedto two different PhP types, not found outside Canada, andtwo of these isolates also had the unique ribotype 13, notpreviously described.

On the basis of PhP typing, a dendrogram of strains ofdifferent geographic origin was generated (Fig. 3). It wasdemonstrated that most of the isolates from Finland, Swedenand Denmark belonged to two major clusters, and most ofthe strains from southern Europe fell into two other distinctclusters. Most isolates from UK, North America, Chile andTasmania grouped together in a distinct cluster. A minorgroup of Finnish isolates clustered together with the Italianisolates belonging to the PhP type IT4. Three of the fiveNorwegian isolates together with one isolate from the USAwere found in one of the major clusters of Finnish, Swedishand Danish isolates, whereas a fourth Norwegian isolate waslocated in the second major cluster of Finnish, Swedish andDanish isolates. The last Norwegian isolate was located inthe cluster of isolates from UK, North America, Chile andTasmania.

Between strains assigned to each PhP type, there are minorvariations that will affect the diversity index. When com-paring PhP type 1 strains from Finland with PhP type IT3strains from Italy, all belonging to serogroup O1 and ribotype1, the Finnish strains showed much higher diversity(Di � 0·51) than the Italian strains (Di � 0·019) (Fig. 4).

DISCUSSION

The V. anguillarum isolates included in the present studywere regarded as representative of the clonal lineages of thisspecies that cause vibriosis in mainly Swedish and Finnishfish farms. In Denmark, the problems with vibriosis are lesspronounced because of effective vaccination of all sea-rearedrainbow trout and turbot prior to transfer to the sea. TheDanish strains of V. anguillarum were therefore collected overa longer period of time than the Finnish and Swedish strains,and also included some non-pathogenic strains isolated fromwater and from the gills, faeces and mucus of healthy rainbowtrout.

The distribution of serogroups with the dominance of O1followed by O2, and the presence of low numbers of non-typeable isolates and isolates belonging to other serogroups,was in accordance with previous reports (Larsen et al. 1994 ;Tiainen et al. 1994). The serological reaction of four strainsthat cross-reacted very strongly with VaNT2 and VaNT4serum was remarkable and has not been reported before, norhas it been observed in our laboratory before, even thoughwe have serotyped thousands of V. anguillarum isolates. How-


ever, in a previous investigation of Finnish V. anguillarumstrains isolated during the period 1989–1991, two strainsreacting only with VaNT2 serum were described (Tiainen etal. 1994). Also remarkable were the facts that each of thesefour strains had a unique ribotype and usually also a uniquePhP type, and that they were isolated on four different farms.This indicates that they cannot be considered as the emerg-ence of a new virulent clone with a serotype not recognizedbefore, but may represent a hitherto unrecognized serogroupof V. anguillarum that, under certain circumstances, may bepathogenic for fish. The pathogenic properties of these strainsare now under further investigation.

Several isolates from Finland carried an 86 kb plasmid.This plasmid was the same as the 90 kb plasmid isolated fromone of the Danish strains and the 86 and 90 kb plasmidsisolated from five Italian strains. This plasmid had previouslybeen described by Larsen and Olsen (1991), Pedersen andLarsen (1995) and Pedersen et al. (1996) among strains fromDenmark, France and Italy, and possibly also from othercountries. However, all of the 13 Finnish isolates carryingthis plasmid belonged to ribotype 4, and 12 of these were ofthe PhP type 2 or the related 2b, whereas the previouslyreported strains from Denmark, France and Italy, that carriedthis 86 kb plasmid, belonged to ribotypes 1, 5 or 6 (Pedersenand Larsen 1995 ; Pedersen et al. 1996). The Finnish strainscarrying the 86 kb plasmid were all isolated in 1993, 1994and 1996 and were not reported in the investigation of strainsfrom 1989–1991 (Tiainen et al. 1994). The combination ofribotype 4, plasmid profile pJM1 plus the 86 kb plasmid, andPhP types 2 or 2b, was only found in Finnish strains, indi-cating that this is a true clonal lineage which was probablynot introduced from outside Finland. Several other examplesof unique combinations of ribotypes, plasmid profiles andPhP types were present, many of which were restricted toone region or even the same fish farm, indicating that thestrains that caused infections were not introduced from out-side the area but were local strains recruited from the environ-ment or other unknown habitats or reservoirs. F. ex., eightserogroup O1 strains from Sweden belonged to the fourunique PhP types, D4, D28, D82 and D99, with two strainsin each group. In two of these groups, the two strains wereisolated on the same fish farm but in two different years,indicating that the strains responsible for the outbreak werenot introduced from outside the farm but may most likelyhave been resident in the area or on the farm. Alternatively,the same clone may have been introduced repeatedly fromthe same external source, e.g. the same hatchery.

The function of the 67 kb pJM1 plasmid is well known. Itwas first shown that this plasmid encodes an iron-seques-tering system consisting of a siderophore, anguibactin, and asiderophore-binding outer membrane protein, OM2 (Crosa1980 ; Crosa and Hodges 1981 ; Actis et al. 1985) ; it was soonunderstood that this system was important for the virulence


Fig. 3 Dendrogram of 72 Vibrio anguillarum serogroup O1 isolates of different geographic origin. NO � Norway, US � USA,SW � Sweden, FL � Finland, DK � Denmark, GE � Germany, IT � Italy, GR � Greece, FR � France, TW � Taiwan,SP � Spain, UK � United Kingdom, TS � Tasmania, CN � Canada and CH � Chile. (ž), Strains from Finland, Sweden andDenmark ; (Ž), strains from the Mediterranean ; (�), strains from other countries. Sal, Rb, Tu, Csa, Sba, Sbr, Mul, Mf, Smand Ssa indicate that the strains were isolated from Atlantic salmon, rainbow trout, turbot, chinook salmon, sea bass, sea bream, mullet,milkfish, Baltic salmon and sockeye salmon, respectively



Fig. 4 Dendrogram of Vibrio anguillarum serogroup O1, ribotype pattern 1 isolates of PhP type 1 from Finland (VF-marked strains),PhP type IT3 from Italy (IT-marked strains), PhP type IT3 from Greece (GR36 and GR35), together with PhP single type from Greece(GR34), three PhP type IT5 strains from Spain (SP-marked strains) and one PhP single type from France (FR40). All strains carriedthe 67 kb pJM1 plasmid. Sba, Sbr, Mul and Tu indicate that the strains were isolated from sea bass, sea bream, mullet andturbot, respectively



of V. anguillarum strains of serogroup O1 (Crosa et al. 1980).Later, it has been demonstrated that the entire iron-seques-tering system is somewhat more complicated, involving geneson the pJM1 plasmid as well as genes on the chromosome(Koster et al. 1991 ; Chen and Crosa 1996 ; Chen et al. 1996).The function of the 86 kb plasmid is at present unknown,but as it is found in strains from several countries and seem-ingly, also in different clonal lineages, this deserves to beinvestigated. It seems, however, not to be involved in viru-lence (Pedersen et al. 1997).

It is known that the pJM1 plasmid exists only in V. anguil-larum strains belonging to serogroup O1 and that the majorityof the virulent strains carry this plasmid (Pedersen and Larsen1995 ; Pedersen et al. 1996). Most of the serogroup O1 isolatesincluded in the present investigation contained pJM1. FiveO1 strains isolated from diseased fish had no plasmids but itis not known whether these strains had the plasmid at thetime of isolation and subsequently lost it during laboratoryprocedures (Pedersen 1997). The 86 kb plasmid has also beenisolated exclusively from O1 strains, and only those strainsas also carried the pJM1 plasmid.

One Finnish isolate carried a plasmid of 73 kb. The normalsize of the pJM1 plasmid is 67 kb, but variations causedby insertions or deletions have previously been described(Pedersen et al. 1996), and it has been demonstrated thatvirulence is not necessarily affected by such variations (Peder-sen et al. 1997). The 73 kb plasmid was shown to be such aderivative of the pJM1 plasmid.

Several isolates of serogroup O2 carried one or two smallcryptic plasmids, varying in size from 4·2 to 7·8 kb, in accord-ance with the observation of Tiainen et al. (1995) who foundthese plasmids only in strains from Scandinavia and notamong strains from southern Europe. The strains belongingto serogroup O2 were considerably more heterogeneous thanserogroup O1 strains, displaying 18 different ribotypes and15 different PhP types among 30 strains. Eight of the 13Swedish O2 strains were from fish species other than rainbowtrout.

Previous investigations of V. anguillarum serogroup O1have so far demonstrated the existence of 12 differentribotypes. Six of these ribotype patterns (2, 7, 8, 9, 10 and 11according to Pedersen and Larsen 1993, 1995) have beenfound in non-pathogenic and environmental strains whereasthe remaining types (1, 3, 4, 5, 6 and 12) have been foundamong fish pathogenic isolates (Pedersen and Larsen 1993,1995 ; Pedersen et al. 1994 ; Skov et al. 1995). Pattern 1 wasshown to be the most prevalent, an observation that is inaccordance with the results of the present study. Pattern 3has previously been demonstrated only in strains from Italy(Pedersen and Larsen 1993, 1995 ; Pedersen et al. 1994 ; Skovet al. 1995), and that observation was confirmed in the presentstudy. Pattern 4 has hitherto only been demonstrated instrains from salmonid fish from the USA and Canada (Peder-


sen and Larsen 1993, 1995 ; Pedersen et al. 1994 ; Skov et al.1995), but the present study showed that this ribotype wasalso prevalent in Sweden and Finland. However, the PhPtypes of these isolates were different from those of the twoCanadian ribotype 4 strains included in this study. Previousstudies have demonstrated the presence of ribotype 6 only inDenmark and Norway (Pedersen and Larsen 1993, 1995 ;Pedersen et al. 1994 ; Skov et al. 1995), but the present studyshowed that this ribotype was also present in Sweden, andmost of these Swedish isolates, as well as the Norwegianribotype 6 isolate, belonged to the same PhP type. TwoCanadian isolates had a ribotype pattern not previously seen.This pattern was designated pattern 13 and these strains alsoshared an identical PhP type not observed in other strains.

Recent investigations using pulsed-field gel electro-phoresis, plasmid analyses and ribotyping have shown thatstrains from northern Europe and southern Europe seem tobelong to different clonal lineages, and that Japanese andAmerican strains are different from these (Skov et al. 1995 ;Tiainen et al. 1995 ; Pedersen et al. 1996). Likewise, in astudy using a combination of different typing methods (Kuhnet al. 1997), it was found that comparing strains from closegeographic locations yielded a lower diversity than comparingstrains from distant locations, and strains from certain fishspecies showed lower diversity than strains from other sour-ces. The present results on the basis of PhP typing confirmedthese observations. As indicated by the dendrogram (Fig. 3),strains from different geographic regions, with few excep-tions, clustered separately.

An interesting observation was that the prevalence of dif-ferent clonal lineages, as judged by their ribotype patterns andPhP types, varied from one year to another and in Finland, itseemed that one clonal lineage, the combination of ribotype 1and PhP type 1, became increasingly important, its prevalenceincreasing from 20% of the isolates in 1992 to approximately80% in 1996. The cause of this development is now knownbut, as previously mentioned, the fact that most combinationsof ribotype and PhP type were restricted to specific geo-graphic areas, together with the fact that the prevalence ofspecific types varies from one year to another, suggests thateither the clones change their properties quickly or they arepresent locally, in some hitherto unknown reservoir, fromwhere they can be recruited and cause infections wheneverthe conditions are right.

The much higher diversity of the Finnish PhP type 1strains compared with the Italian IT3 strains, which were allidentical with respect to serotype, ribotype and plasmidprofile, can be explained by a changing effect ; a specific clonewill develop minor deviations that increase with time. Thus,a develops into b that develops into c, etc., where a and b arevery closely related and b and c are very closely related, buta and c are less closely related. A high diversity index wasalso noticed among other clonal lineages from Finland,


Sweden and Denmark, suggesting that these clonal lineageshave been present there for long enough time to developdescendants that have subsequently developed separately andundergone small evolutionary changes. In contrast, the verylow diversity index among the IT3 isolates shows that thisclone must be a new one that has emerged and spread quicklyand has not yet had time to develop any significant diversity.

The first study of ribotypes of V. anguillarum serogroupO1 was carried out by Pedersen and Larsen (1993). Theseauthors concluded that ribotyping could be a valuable toolin epidemiological investigations of this serogroup. Later, acombination of ribotyping and pulsed-field gel electro-phoresis was found to increase the discriminatory power sig-nificantly (Skov et al. 1995). However, the present studyshowed that ribotyping alone was not sufficiently dis-criminative to distinguish between strains from differentlocations. PhenePlate typing proved to be more dis-criminative and allowed us to follow the evolution of clonesand the development of minor deviations over time, andby using a combination of these two techniques, excellentdiscrimination between strains was obtained. On the basis ofthe results from this study, different typing methods for V.anguillarum are recommended, depending on the collectionof strains as well as the purpose of the study. We wouldcertainly recommend O-serotyping as the primary typingmethod as very important properties, especially virulence,are associated with the O-serogroup. Similarly, we foundimportant information in the plasmid profiles of the strains,especially those belonging to serogroup O1. A more detailedplasmid analysis could have included restriction analysis ofthe plasmids but was not carried out in the present study.For the typing of serogroups other than O1, ribotyping yiel-ded very good discrimination and for those isolates, plasmidsand PhP typing yielded little further information. However,for serogroup O1, ribotyping yielded very limited informationas the vast majority of isolates belonged to only three differentribotypes. For this serogroup, the PhP data were very dis-criminative and provided extremely useful information thatallowed us to draw important conclusions as to the clonality,ecology and epizootiology for this serogroup.

ACKNOWLEDGEMENTS

The technical assistance of Ms Farah Bahrani and the fin-ancial support of The Danish Agricultural and VeterinaryResearch Council, grant no. 9503658, and the Swedish Coun-cil of Forestry and Agricultural Research, grant no.722.0985/93, is gratefully appreciated.

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