Genetic Diversity of the Fish Pathogen Aeromonas Salmonicida Demonstrated by Random Amplified Polymorphic DNA and Pulsed-Field Gel Electrophoresis Analyses

DISEASES OF AQUATIC ORGANISMS Vol. 39: 109-1 19.2000 Published January 14 Dis Aquat Org

Genetic diversity of the fish pathogen Aeromonas salmonicida demonstrated by random amplified polymorphic DNA and pulsed-field gel electrophoresis analyses

Bronagh O'hIcil, Gilles Olivier2,Richard Powelll.*

'Department of Microbiology, National University of Ireland, Galway, Ireland 'Department of Fisheries and Oceans, Gulf Fisheries Centre, PO Box 5030, Moncton, New Brunswick E1C 9K1, Canada

ABSTRACT: The current taxonomy of Aeromonas salmonicida includes 4 subspecies. A. salrnonicida subsp. salmonicida is associated with salrnonid furunculosis, and A. salmon~cidasubsp. achromogenes, A. salmonicida subsp. masoucida, and A. salmonicida subsp. smithia are strains that show variation in some biochenlical properties. This classification does not readly encompass isolates from a wide range of fish hosts currently described as atypical A. salmonic~da.This study examined 17 typical strains, 39 atypical strains and 3 type A. sahonicida subspecies strains for genetic similarity using the random amplified polymorphic DNA (RAPD) and pulsed-field gel electrophoresis (PFGE) techniques. On the basis of RAPD- and PFGE-derived profiles, similarity matrices and dendrograms were constructed. The results showed that species A. salmonicida constituted a genetically heterogeneous group of strains, encompassing within an homogeneous or clonal lineage comprised solely of typical strains and the A. salmonicida subsp. salmonicida type strain.

KEY WORDS: Aeromonas salmonicida . Fish pathogens . Taxonomy . RAPD . PFGE

INTRODUCTION achromogenes (Smith 1963), A. salmonicida subsp. masoucida (Kimura 1969) and A. salmonicida subsp. Aeromonas salmonicida is the bacterial agent associ- smithia (Austin et al. 1989) represent aberrant strains ated with furunculosis, a disease generally confined to that show variation in some biochemical properties. salrnonid fish species (Popoff 1984). The organism is This classification scheme is complicated by 2 obser- readily recovered from the kidney or surface lesions of vations. First, repeated isolations of Aeromonas sal- diseased fish and is conventionally identified by the monicida subsp. masoucida and subsp. smithia have occurrence of dark brown pigmented colonies on tryp- not been reported since their initial description (Wik- tone soya agar after incubation at 20 to 25OC for 2 to lund & Dalsgaard 1998). Second, there have been 4 d. The traditional description of members of this spe- many reports describing isolations of A. salmonicida cies is: non-motile, fermentative, Gram-negative rods from chronic ulcerative diseases found in both sal- which do not grow at 37°C and which produce catalase monid (Evelyn 1971, Paterson et al. 1980) and non- and oxidase (Popoff 1984). The current taxonomy of salmonid species including carp (Bootsma et al. 197?), A. salmonicida includes 4 subspecies; A. saLmonicida minnow (Hastein et al. 1978), goldfish (Elliot & Shotts subsp. salmonicida is associated with salmonid furun- 1980, Whittington et al. 1987), Atlantic cod (Cornick et culosis and has been arbitrarily termed typical to dis- al. 1994), eel (Kitao et al. 1985), sand-eels (Dalsgaard & tinguish this taxon from all other forms of A. salrnoni- Paulsen 1986),pike (Wiklund 1990), flounder (Wiklund cida (Austin & Austin 1993). A. salmonicida subsp. & Bylund 1991, 1993) and turbot (Pedersen et al. 1994). These isolates are termed atypical A. salmonicida and differ in various phenetic traits including colony mor- 'Corresponding author. E-mail: nchard [email protected] phology, growth intensity and temperature range, a

8 Inter-Research 2000 Resale of full article not perrn~tted 110 Dis Aquat Org

requirement for salt (1 % w/v sodium chloride), fer- listed in Table 1. Typical and atypical A. salmonicida mentation of carbohydrates, amlno acid decarboxyla- strains were collected and the strain designations tion, and oxidase reaction (Austin & Austin 1993, Wik- along with host data and location of isolation (where lund & Dalsgaard 1998). Clinical disease signs are known) are also listed in Table 1. All typical strains often characterized by cutaneous ulceration in com- were cultivated in tryptone soya medium (Oxoid, parison to the general septicaemia of typical salrnonid Hampshire, UK) at 2Z°C for 2 d. All atypical strains furunculosis. The significance of this atypical group of were cultivated on either nutrient agar (Oxoid), or A. salmonicida isolates and their expanded host-range columbia base (Oxoid) supplemented with 7 % human with respect to the health of both feral and cultured blood, and incubated at 22°C for between 2 and 5 d. fish stocks is unclear. However, infections by atypical Genomic DNA extraction. Cells were harvested strains are a cause of increasing economic concern in from 10 m1 cultures by centrifugation, 8500 X g for aquaculture (Austin & Austin 1993), and epizootic 10 min (Centrifuge 5416, Eppendorf, Hamburg, Ger- infections of wild fish stocks have also been reported many), and genomic DNA extraction was performed (Wiklund et al. 1994). Finally, their position in terms of using a modification of a previously described method the current taxonomy of A. salmonicida is unresolved. (Ausubel et al. 1992). Briefly, the bacterial pellets were Characterization of genomes is a useful method for resuspended and washed in 1 m1 of sterile H20, fol- both taxonomic and epizootiological studies of bacter- lowed by centrifugation for 5 min at 9650 X g (MSE ial pathogens, and has been applied to Aeromonas sal- Micro Centaur, Sanyo, UK) and resuspension in 400 p1 monicida. DNA:DNA reassociation analysis (Belland & TE buffer (10 mM Tris-HC1, 1 mM EDTA, pH 8.0). The Trust 1988), plasmid profiling (Nielsen et al. 1993), cells were lysed by the addition of 200 p1 of lysozyme ribotyping (Nielsen et al. 1994), DNA probes (Gustaf- (10 mg ml-') (Boehringer Mannheim GmbH, Germany) son et al. 1992, Hiney et al. 1992). restriction endo- and incubation at 37OC for 60 min. The preparation nuclease analysis (McCormick et al. 1990), random was then incubated for 10 min with 40 p1 of Proteinase amplified polymorphic DNA analysis (Hanninen et al. K (10 mg ml-') (Boehringer Mannheim) at room tem- 1995, Miyata et al. 1995), and pulsed-field gel elec- perature, followed by the addition of sodium dodecyl trophoresis (PFGE) (Umelo & Trust 1998) have concen- sulphate (SDS) to a final concentration of 1.0% and trated on A. salmonicida subsp. salmonicida, revealing incubation at room temperature until the preparation this group to be an extremely homogeneous taxon. was clear. For the atypical strains, the preparation was Only the DNA:DNA reassociation (Belland & Trust further incubated at 60°C for 5 min to ensure complete 1988) and PFGE (Umelo & Trust 1998) analyses lysis. Eighty p1 of 0.5 M EDTA were added, mixed by included atypical strains (15 and 16 strains respec- gentle agitation and the solution was deproteinised by tively), and these reports concluded that the atypical sequential phenol and chloroform-isoamyl alcohol strains are genetically diverse when compared to typi- (24:l v/v) extraction. The genomic DNA was precipi- cal strains. tated in ethanol and resuspended in TE buffer. The Here we report a genotypic analysis of 39 strains of DNA concentration was estimated by visual compari- atypical Aeromonas salmonicida in comparison with l? son with standard DNA size markers after electro- typical strains and th.e type strains for 3 A. salmonicida phoresis through a 1% agarose (BioGene Ltd, Cam- subspecies (subsp. smithia was unavailable). Initially, bridge, UK)TAE gel stained with 0.5 pg ml-' ethidium all strains were examined by screening with a putative bromide (Sigma Chemical Co., St Louis, MO, USA). A, salmonicida species-speci.fic oligodeoxynucleotide 16s rRNA gene analysis. The PCR primer desig- DNA probe. Subsequently, the strain collection along nations, sequences, and reference positions on with the type A. salmonicida strains were analyzed alignments of 16s rRNA sequences are EB, 5'- using the random amplified polymorphic DNA (Berg et GAGTTTGATCCTGGCTCAG-3', (bases 3-25) and al. 1994) and PFGE (Schwartz & Cantor 1984) tech- UN, 5'-ACGGNWACCTTGTTACGAGTT-3' (bases niques. Finally, the genetic similarities between both 1423-1402) (standard IUPAC nomenclature: N is G, A, the typical and atypical strains and the diversity en- T, or C; W is A or T). Both PCR primers have been compassed bx species A. salmonicida were evaluated. described as specific for most bacterial species (Lane 1991). The 50 p1 reaction contained 10x KC1 buffer (Biotaq. Bioline Ltd, UK) including 1.5 mM MgCl?, MATERIALS AND METHODS 200 pM dNTPs (Pharmacia, Uppsala, Sweden), 1. U Tag DNA polymerase (Biotaq. Bioline Ltd), 170 pm01 of Aeromonas salmonicida strains. Aeromonas sal- primer EB, 157 pm01 of primer UN and 20 ng of monicida type strains were obtained from the National genomic DNA, overlayed with 30 1-11 of mineral oil. Collection of Industrial and Manne Bacteria (NCIMB), Negative controls containing no target DNA were in- Aberdeen, Scotland, and the strain designations are cluded. The reactions were then amplified using a O'hIci et al.. Genetic diversity of Aeromonas salmonicida 111

TRIO-thermoblock thermocycler (Bio- Table 1. Strains of Aeromonas saln~onicidaexamined in this study In paren metra GmbH, Gottingen, Germany) theses: c, Isolated from cultivated host; W, Isolated from wild host; a, isolate, through 30 cycles of 1 min at 94"C, from host In aquarium; U, host status unknown, A, growth on non-supple mented med~a;B, growth on supplemented media only 1 min at 52°C and 1 min at 72°C followed by a final extension of 5 min at Type strains 72"C, and then held at 15°C. Five p1 XCJ\!B 1102 A. salmonicida subsp, salmonicida aliquots of the amplification products XCILIU 1110 A. salmonicida subsp. achromogenes were analyzed by electrophoresis ?ICIMB 2020 A. salmonlcida subsp. masoucjda through 1 % agarose (BioGene) TAE Typical strains gels stained with 0.5 pg ml-' ethidium Isolate Source P P bromide (Sigma).Slot blot hybridization SRT480 Atlantic salmon Salmo salar. Ireland (c) analysis using the Aeromonas salmoni- 0299/M Atlantic salmon Salmo salar, Ireland (W) cida specific oligodeoxynucleotide BMB2 Atlantic salmon Salmo salar, Scotland (c) (bases 469-486 of A. salmonicida BMA1 Atlantic salmon Saho salar, Scotland (c) MT1014 Brown trout Salmo trutta, Norway (U) subsp. salmonicida 16s rRNA gene) 129/91 Salmonid (species unknown), Norway (U) was performed by end labelling 100 ng 028 Brown trout Saho trutta, USA (U) of the DNA probe, 5'-GGCGCCTAAT- 80204 Atlantic salmon Salrno salar, New Brunswick, Canada (c) SS70.1 Coho salmon Oncorhynchus kitsutch, USA (U) ACGTGTCA-3', with 50 pCi (y-32P) 95063 Atlantic salmon Salnlo salar, Nova Scotia, Canada (c) ATP (DuPont NEN, Hitchin, UK) and BC-? Atlantic salmon Salmo salar, Bntish Columbia, Canada (c) 1 U T4 polynucleotide kinase (Promega, 1018 Atlantic salmon Salmo salar, Quebec, Canada (c) 65-R Salmonid (species unknown), France (U) Madison, WI, USA). 16s rRNA gene 810 Atlantic salmon Salmo salar, Quebec, Canada (c) PCR products were denatured by addi- BJ Atlantic salmon Salmo salar, New Brunswick, Canada (c) tion of a half volume of 1 M NaOH and NS4 Atlantic salmon Salmo salar, Nova Scotia, Canada (c) incubation at 55°C for 5 min followed by A450 Brown trout Salmo trutta, France (U) neutralization by addition of an equal Atypical strains volume of 1 M HC1 prior to application Isolate Soul-ce onto a nylon membrane (Nytran, Schle- M- l (U, A) icher & Schuell, Dassel, Germany) with 86-3 16 LingVasou cod salmon Ophiodon Oncorhynchus elongatus, masou,British Columbia,Japan Canada (W, A) F661-2/89 Atlantic salmon, Salnlo salar, Norway (c, A) a 96-well slot blot apparatus (Schleicher 81377 Atlantic cod Gadus morhua, Canada (W, A) & Schull). Standard hybridization (6x 87048 Atlantic salmon, Salrno salar, New Brunswick,Canada (W, A) (U, SSC, lx Denhardt's solution, 0.1 ",SDS) F1542-2 Atlant~csalmon Salmo salar, Norway A) Brook trout Salvelinus fontinalis, Newfoundland, Canada (W, A) was performed for 8 h at 51°C. The filter K3 88301 Tom cod Gadus microgadus, New Brunswick, Canada (W, A) was then washed twice in 2x SSC for 94504 Tom cod Gadus m~crogadus,New Brunswick, Canada (w, A) 15 min at room temperature, lx SSC for 94326-2 Tom cod Gadus microgadus, New Bruns~nck,Canada (W, A) 15 min at room temperature, and finally 94326-4 Tom cod Gadus m~crogadus,New Brunswick, Canada (W, A) Tom cod Gadus mcrogadus, New Brunswick, Canada (W, A) 0.1~SSC for 15 min at 40°C before 94326-5 Fin3 Brown trout Salmo trutta, Finland (U, A) autoradiography. 143/70 Atlantic salmon Sdho salar, Scotland (U, A) Random amplified polymorphic DNA MT373 Atlantic salmon Salnlo salar, Scotland (U, A) Atlantic salmon Salmo salar, Scotland (U, A) (RAPD). The 3 previously described MT194 MT675 Atlantic salmon Salmo salar, Scotland (U, A) RAPD primers (Caetano-Anolles et al. Atlantic salmon Salmo salar, Newfoundland, Canada (c, B) 88165 1991) used in the study were HI, 5'- Arctic charr Salvelinus alpinus, Newfoundland. Canada (c, B) NFD'95 Atlantic salmon Salmo salar, ricw Brunswick, Canada (c, B) TGCCGAGCTG-3'; H2,5'-AGTCAGC- 87089 Atlantic salmon Salmo salar, Kew Brunswick, Canada (c, B) CAC-3'; and H3, 5'-CGCGCCGG-3'. 86663 Atlantic salmon Salmo salar, Nova Scotia, Canada (W, B) Amplification was performed using a 87480-an Atlantic salmon Salrno salar, Canada (c, B) 87445-1 hot bonnet thermocycler (Omnigene Atlantic salmon Salmo salar, Canada (c, B) 87445-2 Atlantic salrnon Salmo salar, Nova Scotia, Canada TR3 CM220, Hybaid Ltd, Middlesex, 87445 Atlantic salmon Salrno salar, Nova Scotia, Canada UK) with no overlay of mineral oil. The 94450 Eel Angudla anguilla. Nova Scotia, Canada (W, B) 50 p1 reaction contained 10x NH, buffer 91518 Eel Anguilla anguilla, Nova Scotia, Canada (W, B) 91549 Eel Anguilla anguilla, New Brunswick, Canada (W, B) (Bioline Ltd), 4.0 mM MgCl,, 200 pM of 1618.92 Brown trout Salmo trutta, Finland (U, B) dNTPs (Pharmacia), 2 U Taq DNA Poly- Fin5 Unknown host, England (U, B) merase (Bioline Ltd), 60 to 80 pm01 of FP9 Brown trout Salmo trutta, Finland (U, B) Fin6 Brown trout Salmo trutta, Finland (U, B) primer and 20 ng of genomic DNA. Fin? Brown trout Salmo trutta, Finland (U, B) Negative control reaction mixtures Fin8 Brook trout Salvelinus fontinalis, Finland (U, B) lacking DNA template were also in- Fin9 Goldfish Carassisus auratus, USA (a, B) cluded. Amplification conditions were Goldfish Carassjsus auratus, Germany (a, B) Carp Cyprinus carpio, Netherlands (U, B) Flounder Platichthys flesus, Finland (W, B) 112 Dis Aquat Org 39: 109-119, 2000

35 cycles of 10 S at 94OC, 30 s at 37"C, 60 S at 72OC and RESULTS a final extension of 5 min at 72°C. Five p1 aliquots of the amplification products were analyzed by elec- 16s rRNA gene analysis trophoresis through 1 % agarose (BioGene), 1 % NuSieve (FMC Bioproducts, Rockland, ME, USA) TAE Initially, to confirm that all the atypical strains were gels stained with 0.5 pg ml-' ethidium bromide and members of the species Aeromonas salmonicida, their visualized over UV using the Bio-Profil imaging system 16s rRNA encoding genes were amplified by PCR and (Vilber Lourmat, Marne La Vallee, France). The 100 bp the amplification products were screened with an DNA ladder (Life Technologies, Gibco BRL, Renfrew- oligodeoxynucleotide DNA probe designed to specifi- shire, UK) standard length DNA markers were cally target the species A. salmonicida. Nucleotide included in each electrophoresis. sequences have been described for the 16s rRNA Pulsed field gel electrophoreses (PFGE). Colonies of genes of A. salmonicida subsp, salmonicida (GenBank each strain were diluted in sterile H20 until an optical accession number X7 1836), subsp. achromogenes density (OD) at 600 nm of approximately 0.1 was (X60407), subsp. masoucida (X74680), subsp. smithia reached. Genomic DNA was prepared in chromosomal (AJ009859) along with the related aeromonad species, grade ultra-pure DNA agarose (Bio-Rad Laboratories, A. hydrophila (X74677), A. sobria (X74683), A. media Richmond, USA) as previously described (Cameron et (X74679), and Haemophilus piscium (AJ009860) (Mar- al. 1994). Digestion with restriction enzymes was per- tinez-Murcia et al. 1992, Thornton et al. 1999). A DNA formed by cutting a slice from 1 DNA-containing sequence alignment was performed using these agarose plug and incubating for 1 h in the relevant sequences and an 18 base oligodeoxynucleotide DNA restriction enzyme reaction buffer followed by 4 h with probe was designed complementary to bases 469 to 20 U of restriction enzyme. SpeI enzyme and reaction 486 of the A. salmonicida subsp. salmonicida 16s rRNA buffer were obtained from New England Biolabs sequence. Under the appropriate hybridization condi- (Hertfordshire, UK) and XbaI enzyme and reaction tions, this DNA probe will only recognise the 16s buffer were obtained from Promega. The DNA frag- rDNA sequences of the 4 accredited A. salmonicida ments were then subjected to electrophoresis in a 1 % SeaKem agarose (FMC BioProducts) gel submerged in 0.5~Tris-borate-EDTA buffer using a modified contour clamped homogenous electric field (Pulsaphor Plus, Pharmacia). The running conditions were 12 V cm-' at 14°C for 22 h. The pulse times were increased by step- ping as follows: 2 s for 5 h, 5 s for 6 h, 9 s for 6 h, and 12 s for 5 h for XbaI digestions; and 5 s for 3 h, 9 S for 5 h, 12 S for 5 h, 20 s for 4 h, 25 S for 3 h and 30 s for 2 h for SpeI digestions. The concatenated lambda DNA ladder (Pharmacia) was included in each electrophoresis. After electrophoresis, gels were stained in 2 pg ml-' ethidum bromide for 15 min, destained in distilled H20for 15 min, and visualized over UV using the Bio- Profil imaging system (Vilber Lourmat). RAPD and PFGE cluster analysis. The molecular lengths of the RAPD- and PFGE-generated DNA fragments were determined using ihe Bio-Gene software package (Vilber Lourmat) on the Bio-Profil imaging system. Each strain was then visually coded for the Fig. 1. Aeromonas salmonicida specific 16s rDNA slot blot presence (coded 1) or absence (coded 0) of each DNA hybridization. Row A: 1, M-l; 2, 86-316; 3, F661-2/89; 4, fragment. The data obtained for all 3 RAPD primers 81377; 5, 87048; 6, F1542-2; 7, K3; 8, 88301; 9, 94504; 10, were combined into 1 data matrix which was then ana- 94326-2. Row B: 1, 94326-4; 2. 94326-5; 3, Fin3; 4, 143/70; 5, MT373; 6. MT194; 7. MT675; 8, 88165; 9, NFD'95; 10, 87089. lyzed using the inverse of Jaccard's similarity coeffi- Row C: 1,86663; 2,87480-an; 3,87445-1; 4,87445-2; 5,87445; cient as previously d.escribed (Morgan et al. 1993). A 6. 94450; 7, 91518; 8, 91549, 9, 1618.92, 10, Fin5 Row D: 1, similar analysis was performed on the PFGE profiles FP9; 2, Fin6; 3, Fin7; 4, Fin8; 5, Fin9; 7, 3.111; 8, 82-83; 9, generated by both SpeI and XbaI restriction enzymes. V234/81; 10, T5. Row E: 1, A. sobria; 2, NCIMB 1102; 3, NCIMB 1110; 4, NCIrMB 2020; 5, A. media; 6, A. hydrophila; The similarity dendrograms were generated using the 7, E. coh. Note: Row D, 6 represents 1 atypical strain that was UPGMA clustering method of the PHYLIP phylogeny later removed from this study due to subsequent bacterial inference package (Felsenstein 1993). contamination of stocks O'hIci et al.: Genetic diversity of Aeromonas salrnonicida 113

subspecies and H. piscium and should not react with combined, no 2 atypical strains produced an identical other aeromonads. Fig. 1 shows that the 16s rDNA RAPD DNA profile to each other, although similar amplification products from the atypical A. salmoni- RAPD DNA profiles among some atypical strains could cida strains (Fig. 1, Rows A, B, C & D) along with the be seen with individual RAPD primers (Fig. 3A, lanes 9 type strains A. salmonicida subsp. salmonicida, A. & 10 or lanes 13 & 14; 3B, lanes 7 & 8, lanes 15 & 16, or salmonicida subsp. achromogenes, and A. salmonicida lanes 17 to 21; 3C, lanes 19 & 20). While primer H2 subsp. masoucida (Fig. 1, Row E, Columns 2, 3 & 4 resulted in the greatest number of common DNA frag- respectively), were recognised by this DNA probe. ments between various atypical strains, primer H3 pro- Other species such as A. media, A. hydrophila and E. duced individual DNA patterns lvith each strain with coli showed no hybndization (Fig. l., Row E, Colun~ns few common DNA fragments. 5, 6 & 7 respectively), and A. sobria revealed only a The lengths of the DNA fragments generated by the low-intensity hybridization signal (Fig. 1, Row E, Col- RAPD primers were determined and the combined umn 1). Bases 469-486 of the 16s rDNA of A. sobria RAPD profiles of the typical, atypical and type strains are the most similar in sequence homology to the were resolved into numerical matrices and subsequent respective region of the A. salmonicida 16s rDNA with dendrograms describing genetic similarity. Fig. 4 only 2 variant bases. All the atypical A. salmonicida shows the RAPD-based similarity dendrogram pre- strains analyzed in this study reacted with this A. pared for the typical Aeromonas salmonicida strains. salmonicida species-specific DNA probe providing evidence for their membership of this species.

RAPD analysis

Using the RAPD conditions described, the 3 RAPD primers reproducibly amplified 100 DNA fragments that could be scored for similarity between the Aero- monas salrnonicida strains. Fig. 2 shows the RAPD DNA profiles produced for the typical A. salmonicida strains using RAPD primer H1 (Fig 2A), RAPD primer H2 (Fig. 2B) and RAPD primer H3 (Fig. 2C). None of the 3 type A. salmonicida subspecies strains showed a pattern identical to one of the other strains with any of the 3 RAPD primers (Fig. 2, lanes 2, 3 & 4 in gels A, B, & C) although almost all the RAPD fragments amplified from A. salmonicida subsp. masoucida NCIMB 2020 (Fig. 2, lane 4) were contained within the RAPD profile of A. salmonicida subsp. salmonicida NCIMB 1102 (Fig. 2, lane 2). The RAPD profiles amplified from the typical A, salmonicida strains were clearly similar to one another with both the H1 and H2 primers, while differences between some typical strains could be noted with the H3 primer (Fig. 2, lanes 5 to 21 in gels A, B, & C). The RAPD profiles of these typical strains produced by primers H1 and H2 were very similar to those amplified from the type A. salmonicida subsp. salmonicida strain (Fig. 2A, lane 2; and Fig. 2B, lane 2). Fig. 3 shows the RAPD DNA profiles produced for many of the atypical Aeromonas salmonicida strains using RAPD primer H1 (Fig. 3A), RAPD primer H2 Fig. 2. RAPD profiles of typical Aeromonas salmonicida (Fig. 3B) and RAPD primer H3 (Fig. 3C). It was readily strains. (A) RAPD primer HI; (B) RAPD primer H2; (C) RAPD apparent that, regardless of the RAPD primer used, Primer H3. Lanes: 1, 100 bp DNA molecular size markers none of the atypical strains produced a RAPD DNA (800 bp marker noted on the left); 2, NCIMB 1102; 3. NCIMB 1110; 4, NCIMB 2020; 5, 0229/M; 6, SRT480; 7, MT1014; A. profile similar to any of the type salmonicida sub- 8, 129/91; 9, BMB2; 10, BMAI; 11, 028; 12, BC-7; 13, 1018; species strains (Fig. 3, lanes 2, 3 & 4 in gels A, B, & C). 14, BJ; 15, SS70.1; 16, NS4; l?, 80204; 18, 95063; 19, 65-R; Also, when the results of all 3 RAPD primers were 20, 810; 21, A450 114 Dis Aquat Org 39: 109-119, 2000

Fig. 5 shows the RAPD-based similarity dendrograms prepared for the atypical Aeromonas salmonicida strains. By comparison with the typical strains, genetic heterogeneity among the atypical strains was readily apparent with 23 of 39 atypical strains showing less than 70% RAPD-based similarity to their closest neighbour. No atypical strain showed a 70% or greater RAPD-based similarity to any of the 3 type A. salmonicida subspecies strains. The only well-defined cluster contained 4 atypical strains (143/70, MT67.5, MT373 and MT194) isolated from Atlantic salmon in Scotland. 10 12 13 14 16 17 18 19 20 21 B PFGEanalysis

Initially, 10 atypical Aeromonas salmonicida isolates 800 - were screened by PFGE using the enzymes NotI, XhoI, XbaI and SpeI to determine which restriction endonu-

I I SS70.1 I

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 2021 I I -1 NS4 C Il I BJ I I I 80204 I I I 95063 800- I I I 1018 I 7 I I 65-R I I 810 I Fig. 3. RAPD profiles of atypical Aeromonas salmonicida 1- l A450 strains. (A) RAPD primer HI; (B)RAPD primer H2; (C) RAPD l Primer H3. Lanes. 1, 100 bp DNA molecular size markers (800 I I - BC-7 bp marker noted on the left); 2. NCIMB 1102; 3, NCIMB 11 10; I I 4, NCIMB 2020; 5, M-l; 6, 86-316; 7, F661-2/89; 8, 81377; 9. l NCIMB 1102 87048; 10, F1542-2; 11, K3; 12, 88301; 13, 94504; 14, 94326-2; 15, 94326-4; 16, 94326-5; 17, Fin3; 18, 143/70; 19, MT373; 0229m 20, MT194, 21, MT675 - - SRT480 BMB2 The most distantly related strain was the type A. - BMAl salmonicida subsp. achromogenes strain (NCIMB 1 l lO),which showed less than 70% RAPD-based sln11- 12919I larity to all other typical and type strains. The remain- MT1014 ing strains were separated into 2 clusters which bifur- cated at a point just greater than 70% RAPD-based 028

similarity. The type A, salmonicida subsp. salmonicida NCIMB 2020 strain (NCIMB 1102) formed a cluster which, with the

I NCIMB 1110 exception of the French isolates, strains 65-R and I l A4.50, contained 8 typical strains isolated in North I I America. The type A, salmonicida subsp. masoucida 706 strain (NCIMB 2020) formed a cluster which, with the Fig. 4. UPGMA dendrogram describing RAPD-based genetic exception of strain 028 (isolated from brown trout in similarity of typical Aeromonas saho~cida A vertical the USA) contained 6 typical Strains isolated in north- Line denoting a hypothetical node of 70% RAPD-based simi- ern Europe. larity is added for comparative analysis O'hIcl et al.. Genetic diversity of Aeromonas saln~onicida 115

l I contained sufficient endogenous nucleases to l NCIMB 1102 NFD'95 degrade the genomic DNA within the agarose I I I NClMB 2020 plugs prepared for PFGE. Fig. 6 shows the PFGE DNA profiles produced for the type and typical Aeromonas salmonlcida strains using the restriction endonucleases XbaI (Fig. 6A) and SpeI (Fig. 6B). In a similar manner to the RAPD analysis, none of the 3 type A. salmonicida subspecies strains showed an identical PFGE pattern to one another (Fig. 6A, lanes 2, 3 & 4) although A. salmonicida subsp. masou- ada (NCIMB 2020) shared many similar-length PFGE DNA fragments with A, salmonicida subsp. salmonicida (NCIMB 1102). The PFGE profiles found among the typical strains were very similar to one another and identical among the majority of strains (Fig. 6A, lanes 5 to 20; and Fig. 6B, lanes 2 to 18). This PFGE profile was also clearly similar to that of the type A. salmonicida subsp. salmonicida strain (Fig. 6A, lane 2). Fig. 7 shows the PFGE DNA profiles produced for many of the atypical A. salmonicida strains using the restriction endonucleases XbaI (Fig. ?A) and SpeI (Fig. TB). Only 2 atypical strains, 87445 and 87445-2, produced an identical PFGE DNA profile with both XbaI and SpeI. Very similar, but not identical, PFGE DNA profiles could be seen among some of the atypical strains (Fig. ?A, lanes 7, 8, 9 & 10 or lanes l l, I 3.111 12 & 13; Fig. 7B, lanes 7, 8, 9, 10 & 11 or lanes I 82-83 I TS 12, 13 & 14). I l NC~MB1 I 10 Cluster analysis was performed using the I PFGE data in a similar manner to the analysis of 76 % 0.01 the RAPD data. However, no analysis was per- Fig. 5. UPGMA dendrogram describing RAPD-based genetic simi- formed on the typical Aeromonas salmonicida larity of atypical Aeromonas salmonicida strains. A vertical line strains as there was insufficient variation among denoting a hypothetical node of 70% RAPD-based similarity is the strain profiles, i.e. all the typical strains added for comparative analysis showed a 90% or greater similarity to each other. With respect to the atypical strains, only cleases produced the most informative DNA profiles DNA fragments greater than 100 kb in length were for strain comparison. Not1 and XhoI digestion resulted scored in this analysis. Using both XbaI and SpeI in greater than 30 DNA fragments of less than 150 kb restriction endonucleases, 50 DNA fragments were in length, XbaI digestion resulted in approximately 40 scored for similarity between the atypical A. salmoni- DNA fragments of between 20 and 250 kb in length, cida strains. Fig. 8 shows the PFGE-based similarity and SpeI digestion resulted in approximately 30 DNA dendrogram prepared for the atypical A. salmonicida fragments of between 20 and 500 kb in length. Based strains. As with the RAPD analysis, genetic hetero- on these results, the endonucleases XbaI and SpeI geneity anlong the atypical strains was readily appar- were selected for analysis of genome similarity among ent with 21 of 32 atypical strains showing less than the A. salmonicida strains. Subsequently, however, 70% PFGE-based similarity to their closest neighbour. restriction endonuclease and PFGE analysis could not No atypical strain showed a 70% or greater PFGE- be performed on 6 of the atypical strains (strains 94504, based similarity to the type A. salmonicida subsp. K3, 94326-4, T5, 86663 and 94450) as the genomic salmonicida (NCIMB 1102) or subsp. achromogenes DNA was severly degraded. Despite repeated at- (NCIMB 1110) strains, while 3 atypical strains from tempts, this problem could not be solved and it may be Finland showed a 75% PFGE-based similarity to the that these particular atypical A. salmonicida strains type subsp, masoucida (NCIMB 2020) strain. 116 Dis Aquat Org 39: 109-1 19,2000

strains reacted positively with this DNA probe reflect- ing a common genetic relationship between these members of the species A. salmonicida. To further resolve this relationship, the strain collection was examined using the RAPD and PFGE techniques. DNA profiling using the RAPD technique has been shown to be a very useful method for discriminating among strains of a species (Berg et al. 1994). It has been used successfully to demonstrate genetic heterogeneity among field isolates of Mycoplasma hyopneumoniae

Fig. 6. PFGE profiles of typical Aeromonas salmonicida strains. (A) Xbal digestions. Lanes: 1 & 22, concatenated phage lambda DNA (48.5 kb) molecular size markers (97 kb marker noted on the left); 2, NCIMB 1102; 3, NCIMB 1110; 4, NCIMB 2020; 5, SRT480; 6, 0299/M; 7, BMB2; 8, BMA1; 9, 129/91; 10, MT1014; 11, 028; 12, BJ; 13, 1018; 14, BC-?; 15, 65- R;16, 80204; 17, SS70 1; 18, 95063; 19, NS4; 20, 810; 21, A450. (B) SpeI digestions, Lanes 1 & 22, concatenated phage lambda DNA (48.5 kb) molecular size markers (97 kb marker noted on the left); 2, SRT480; 3.0229/M; 4, BMB2; 5, BMA1; 6. 129/91; 7, MT1014; 8, 028; 9, BJ; 10, 1018; 11, BC-?; 12, 65-R; 13, 80204; 14, SS70.1; 15, 95063; 16, NS4; 17, 810; 18, A450; 19, blank; 20 & 21, non-aeromonad strains

Fig. 7. PFGE profiles of atypical Aeromonas salmonicida strains. (A) XbaI digestions, Lanes: 1, concatenated phage DISCUSSION lambda DNA (48.5 kb) molecular size markers (97 kb marker noted on the left); 2, NCIMB 1102; 3, NCIMB 11 10; 4, NCIMB This study describes a genotypic analysis of 17 typi- 2020; 5, 88165; 6, NFD'95; 7, 87089; 8, 87480-an; 9, 87445-1; 10, 87445-2; 11, 87445; 12, 91518; 13, 91549; 14, 1618.92; 15, cal and 39 atypical strains of Aeromonas salmonicida Fin5; 16, FP9; 17, Fin6; 18, Fin7; 19, Fin8; 20, Fin9, 21, 3 111; isolated from a variety of host species and geographic 22, 82-83; 23, V234/81. (B) SpeI digestions, Lanes: 1, concate- locations and also included type strains representing 3 nated phage lambda DNA (48.5 kb) molecular size markers A. salmonicida subspecies. Initially, all the atypical (97 kb marker noted on the left); 2, NCIMB 1102; 3. NCIMB strains were screened with an oligodeoxynucleotide 1110; 4, NCIMB 2020; 5,88165; 6, NFD'95; 7,87089; 8,87480- an; 9, 87445-1; 10. 87445-2; 11, 91518; 12, 91549; 13, 1618.92; DNA probe designed to specifically recognise mem- 14, Fin5; 15, FP9; 16, Fin6; 17, Fin7; 18, Fin8; 19, Fin9; bers of the species A. salmonicida. All the atypical 20, 3.111; 21, 82-83; 22, V234/81; 23, T5 O'hIci et al.. Genetic diversity of Aeromonas salinonicida 117 --

sible to resolve the strains into 2 clusters within which the majority of the strains correlated with the geographic area of isolation, i.e. one cluster contained predominantly North Ameri- can strains (8 of 10 strains) and the second cluster contained predominantly northern Eu- ropean strains (6 of 7 strains). With respect to the atypical A. salmonicida strains examined, the RAPD analysis highlighted their genetically heterogeneous nature, with none of the atypical strains showing any close similarity with the type A. salmonicida subspecies strains. Within the atypical strains, little clustering of strains correlating with fish host and geographical location were found. The only 2 I 1 I I NCIMB 11 10 correlations of note were strains 143/70, MT675, MT373 and MT194, whch were all isolated from Atlantic salmon in Scotland, and strains 91518 and 91549, which were isolated from eel in Nova Scotia. The only other eel iso- ! I NClMB 2020 lation in the collection, strain 1618-92 from New Brunswick, was placed as their closest neighbour although at less than 70% RAPD- based similarity. In a similar fashion to RAPD analysis, PFGE of large genomic DNA fragments has been shown to be a valuable typing method for epidemiological investigations (Lin & Johnson 1995), including clonal studies (Skov et al. 1995), and has proved to be highly differentiat- ing when other methods were not suitable (Wong et al. 1996). In this study, the PFGE analysis using 2 restriction endonucleases showed the typical Aeromonas salmonicida Fig. 8. UPGMA dendrogran~describing PFGE-based genetic sirni- strains to have greater than 90% similarity to laity of the atypical ero or salmonicidanon as strains. vertical line one another and also to the type A. salmoni- denoting a hypothetical node of 70% PFGE-based similarity is cida subsp. salmonicida straln (NCIMB 1102). added for comparative analysis This data supported the results found from I- CeuI digestion and subsequent PFGE analysis (Artiushin & Minion 1996), to differentiate strains of of 9 typical A. salmoniclda strains reported previously Salmonella enteritidis (Lin et al. 1996) and to examine (Umelo & Trust 1998). With respect to the atypical reports of clonal lineages of Pseudomonas aeruginosa strains examined, the PFGE analysis also highlighted associated with human disease (Mahenthiralingam et their genetic heterogeneity. In this case, the noteworthy al. 1996).One previous study describing RAPD analysis correlations with strain provenance included the clus- of 13 typical Aeromonas salmonicida subsp. salmoni- tering of the 3 Canadian eel isolates, 91518, 91549 and cida strains isolated from the UK, USA and Japan found 1618-92, and a cluster of Canadian Atlantic salmon iso- identical RAPD profiles among all the typical strains lates, 87089, 87480-an, 87445-1 and 87445-2. (Miyata et al. 1995). A second RAPD study of typical A. While both the RAPD and PFGE analyses showed salmonicida strains also found very homogeneous pat- the close genetic sundarity of typical strains and Aero- terns with few polymorphic loci (Hanninen et al. 1995). monas salmonicida subsp. salmonicida, and the het- In this study, the use of 3 RAPD primers that amplified erogeneity present among atypical strains, there was 100 DNA fragments for use in strain comparison also little correlation between the 2 methods in identifying showed the close genetic relationship between the typ- clusters of similar atypical strains. Primarily, this is ical A. salmonicida strains. However, unlike the previ- likely due to the small number of strains examined and ous RAPD studies of typical A. salmonicida, it was pos- the large genetic variation detected by both methods 118 Dis Aquat Org 39: 109-119. 2000

in this study. Also, discrepancies between strain typing Acknotuledgements. We are grateful to colleagues for the by RAPD and PFGE have been reported for other bac- gifts of strains, and the support of the E.U. AIR Programme (AIR3-CT94-1884) and BioResearch Ireland. B.OfhI.was sup- teria (Struelens et al. 1993, Barbier et al. 1996), and ported by NUIG and Enterprise Ireland post-graduate fellow- may be explained by the fact that these methods ex- ships, and is grateful to the European Molecular Biology plore different types of DNA polymorphisms. PFGE is Organisation (EMBO), and J. L. Larsen, M. N. Skov and K. based on restriction enzyme polymorphisms, while Pedersen of the Royal Vetennary and Agricultural University, RAPD analyzes sequence polymorphisms of regions Frederiksberg, Denmark, for their help with PFGE. complementary to the amplification primers and also length polymorphisms of the regions amplified. Fur- LITERATURE CITED thermore, it has been reported that plasmids represent approximately 20% of the A. salrnonicida genome Artiushin S, Minion FC (1996) Arbitrarily primed PCR analysis of ~Mycoplasma hyopneumoniae field isolates demon- (Belland & Trust 1988) although less information is strates genetic heterogeniety. Int J Syst Bacteriol 46: available on the plasrnid content of atypical A. 324-328 salmoniclda strains. These endogenous plasmids may Austin B, Austin DA (1993) Aeromonadaceae representatives act as targets for RAPD primers, resulting in the ampli- (Aeromonas salmonicida). In: Austin B. Austin DA (eds) fication of DNA fragments that would interfere with Bacterial fish pathogens: disease in farmed and wild fish, 2nd edn. Ellis Horwood Ltd, Chichester, p 86-170 the similarity analysis. In this analysis, we sought to re- Austin B, Austln DA, Dalsgaard I, Gudmundsdottir BK, Hoie move interference by plasmids within the PFGE analy- S, Thornton JM. Larsen JL, O'hIci B, Powell R (1998) Char- sis by analyzing onl.17 DNA fragments greater than acterisation of atypical Aeromonas salmonicida by differ- 100 kb in length. However, no such limitation could be ent methods. Syst Appl Microbiol21:50-64 made on the RAPD analysis. This may explain some of Austin DA, McIntosh D, Austin B (1989) Taxonomy of fish associated Aeromonas spp., with the description of Aero- the differences found between the RAPD and PFGE monas salmonicida subsp. smithia subsp. nov. Syst Appl strain clusters, and it shows that molecular typing Microbiol 11:277-290 methods may have some of the disadvantages, includ- Ausubel FM. Brent R, Gngston RE, Moore DD, Seidman JG, ing subjectivity, associated with strain characterisation Smith JA, Struhl K (1992) Short protocols in molecular biology, 2nd rdn. John Wiley & Sons, New York using phenetic traits. These problems have previously Barbier N, Saulnier P, Chachaty E, Dumontier S, Andremont been noted in studies of atypical A. salmonicida A (1996) Random amplified polymorphic DNA typing ver- (Austin et al. 1998). In this study, the RAPD technique sus pulsed-field gel electrophoresis for epidemiological produced 100 loci for strain comparison, which was typing of vancomycin-resistant enterococci. J Clln Micro- twice the number of scorable loci produced by PFGE. biol 34:1096-1099 Belland RJ. Trust TJ (1988) DNA:DNA reassociation analysis Based on the results, modifications of both the RAPD of Aeromonas salmonicida. J Gen Microbiol. 134:307-315 and PFGE conditions or other genotype methods pro- Berg DE, Akopyants NS, Kersulyte D (1994) Fingerprinting ducing a lower number of comparable loci may be microbial genomes using the RAPD or AP-PCR method. more suitable for the identification of strain clusters of Meth Mol Cell Biol 5:13-24 Bootsma R, Fijan N, Blommaert J (1977) Isolation and prelim- epizootiological significance among the atypical A. inary identification of the causative agent of carp erythro- salrnonicida strains. Also, in terms of technique su~t- dermatitis. Vet Arch 47:291-302 ability or robustness, problems were experienced with Boyd EF, Hiney MP, Peden JF, Smith PR (1994) Assessment of both techniques. Reproducibility of DNA profiles using genetic diversity among Aeromonas salmonicida isolates the RAPD technique proved onerous if different ther- by multi enzyme electrophoresis. J Fish Dis 17:97-98 Caetano-Anolles G, Bassam BJ, Gresshoff PM (1991) DNA mocylcers were used, and some of the atypical stralns amplification fingerprinting using very short arbitrary could not be profiled with the PFGE technique. oligonucleot~deprimers. Biotechnology 9553-557 Finally, in terms of population structure, one previous Cameron DN. Khambaty FM, Wachsmuth IK, Tauxe RV. Bar- study using multilocus enzyme electrophoretic analysis ret TJ (!P94) Molecular cha:acterization of Vibrio cholera 01 strains by pulsed-field gel electrophoresis. J Clin differentiated Aeromonas salmonicida into 2 clones Microbiol 32 1685-1690 (Royd et al. 1994), one containing typical A. salmoni- Cornick JW, hlorrison CM, Zwicker B, Shum G 11994) Atypi- cida strains and the other containing atypical strains. cal Aeromonas salmonicida infection in Atlantic cod, However, the RAPD and PFGE analyses showed that Gadus morhua. 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Editorial responsibility: Larry Vauyhan, Submitted: June 13, 1999; Accepted: November 21, 1999 Portsmouth, New Hampshire, USA Proofs received from author(s): January 6, 2000