Molecular analysis of Flavobacterium psychrophilum isolates from salmonid farms in Chile

Pulsed-field gel electrophoresis (PFGE) using different restriction enzymes was used for examining the genetic variability within Chilean Flavobacterium psychrophilum field strains isolated from farmed Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss). Clustering of the SacI restriction patterns distinguished the Chilean isolates in two genetic groups that were strongly related to the host origin of the isolates (trout or salmon). When the Dice coefficient was applied the similarity between the two clusters was of 87.5%. Restriction digest patterns resolved by endonucleases BlnI and XhoI did not differentiate the Chilean F. psychrophilum isolates as shown with SacI; trout isolates were clustered together with Atlantic salmon strains. An average band similarity of 83.2 ± 1.3% was found between all Chilean F. psychrophilum isolates with both endonucleases. PFGE, regardless of the endonuclease employed, was able to clearly distinguish Chilean F. psychrophilum from the type strain ATCC 49418T and the Scottish isolate B97026. The results obtained in this work corroborate the existence of a great similarity within this group of Chilean F. psychrophilum strains, indicating that the disease outbreaks in Chilean farms are dominated by a closely related cluster of strains. Flavobacterium psychrophilum (formerly Cytophaga psychrophila and Flexibacter psychrophilus) is the causative agent of bacterial cold-water disease (BCWD) in a large number of freshwater fish species worldwide (see review Nematollahi et al., 2003) and is of considerable economic significance to aquaculture producers. In Chile, F. psychrophilum was first isolated from a rainbow trout (Oncorhynchus mykiss) farm in 1993 (Bustos et al., 1995); subsequently, this pathogen has been detected in other host species, such as Atlantic salmon (Salmo salar) and Coho salmon (Oncorhynchus kisutch) in different regions at the south of the country. In general, disease outbreaks occurred repeatedly during winter months (water temperatures lower than 12oC) in the freshwater stage of husbandry causing serious losses to Chilean salmonid farms, where infections can result in 5% to 70% cumulative mortality rate of the affected population. A frequent practice to control bacterial pathologies in Chilean salmonid culture is the intensive use of Bull. Eur. Ass. Fish Pathol., 29(6) 2009, 185 antimicrobial agents, mainly oxytetracycline (Miranda & Zemelman, 2002), which is very effective in reducing BCWD induced mortality. We have characterized F. psychrophilum isolates derived from several outbreaks in Chile using phenotypic, antigenic and genetic techniques, including the randomly amplified polymorphic DNA (RAPD-PCR), enterobacterial repetitive intergenic consensus sequence PCR (ERIC-PCR) and repetitive extragenic palindromic PCR (REP-PCR) (Valdebenito & Avendaño-Herrera, 2009). All genetic procedures indicated homogeneity among the Chilean F. psychrophilum strains studied, which was very distinct from the type strain ATCC 49418T isolated from Oncorhynchus kisutch in the USA. To better assess genetic diversity we applied pulsedfield gel electrophoresis (PFGE) in the present study. This technique has been successfully employed to evaluate genetic variability in other fish and shellfish pathogens such as Vibrio anguillarum (Skov et al., 1995), Aeromonas salmonicida (Hänninen & Hirvelä-Koski, 1999; O’hIci et al., 2000), Streptococcus iniae (Fuller et al., 2001), Photobacterium damselae ssp. piscicida (Kijima-Tanaka et al., 2007), Vibrio alginolyticus (Ren et al., 2008), Vibrio ordalii (Silva-Rubio et al., 2008), Streptococcus phocae (Valdés et al., 2009) and Flavobacterium columnare (Soto et al., 2008). Arai et al., (2007) used PFGE with the endonuclease BlnI and XhoI and reported diversity of Japanese F. psychrophilum isolates from Ayu (Plecoglossus altivelis) and other fish, and found evidence for host-specific association between clonal types. More recently, Chen et al., (2008) also employed PFGE with American F. psychrophilum isolates, but used the enzyme SacI, and observed higher similarity in PFGE pattern among rainbow trout isolates relative to Coho salmon isolates. In this work, we investigated the genetic variability within Chilean field strains isolated from Atlantic salmon and rainbow trout by PFGE, with the aim of evaluating this technique as an epidemiological tool. In addition, our PFGE utilized all endonucleases previously used by other researchers for typing F. psychrophilum. Fourteen F. psychrophilum isolates were examined in this study (Table 1). This collection comprised 12 Chilean strains isolated from Atlantic salmon and rainbow trout, which belongs to the different serological groups described in this country (Valdebenito & Avendaño-Herrera, 2009), strain B97026 was isolated from rainbow trout in UK (Faruk et al., 2002; Vatsos et al., 2002) and type strain ATCC 49418T. Their identification and characterization has been confirmed as F. psychrophilum species previously by Valdebenito & Avendaño-Herrera (2009). For all experiments, the strains were routinely grown on TYES agar plates (tryptone yeast extract salts medium: 0.4% tryptone, 0.05% yeast extract, 0.02% anhydrous calcium chloride, 0.05% magnesium sulphate heptahydrate, pH 7.2) or TYES broth (without 1% bacteriological agar) and incubated aerobically at 15 oC for 3–5 days. Stock cultures were maintained frozen at – 80oC in Criobilles tubes (AES Laboratory). Single F. psychrophilum colonies grown on TYES agar were suspended with sterile cotton swabs in 5 ml of TYES broth, and incubated Bull. Eur. Ass. Fish Pathol., 29(6) 2009, 186 in a shaking incubator for 30 h at 15oC to obtain a final density of 1.0 at 600 nm. To overcome DNA degradation problems caused by extracellular DNases, F. psychrophilum cells were fixed with formaldehyde (Soto et al., 2008). Briefly, cells were harvested by centrifugation at 10,000 × g for 10 min at 4oC. The supernatant was discarded, and bacteria were washed twice with 5 ml of PIV (10 mM Tris [pH 7.5], 1 M NaCl) containing 4.0% (v/v) formaldehyde solution and suspended in the same buffer. The mixture was incubated on ice for 1 h. To obtain complete lyses, the cells were washed twice with PIV. Then, 200 μl of the cells suspension was blended into 200 μl of 1% SeaKem Gold Agarose and 8 μl of proteinase K (20 mg ml-1). The cell-agarose mixture was immediately poured into appropriate plug mould and allowed to solidify for 20 min at room temperature. Plugs were transferred to 15 ml tubes each containing 5 ES buffer (50 mM Tris, 50 mM EDTA [pH 8.0]; 1% sodium lauryl sarcosine) and 25 μl of proteinase K for 2 h at 54oC with gentle shaking. The plugs were rinsed three times for 15 min each with preheated 10 ml of sterile distilled water. After the final wash, plugs were transferred into tubes containing TE (100 mM Tris, pH 8.0; 100 mM EDTA, pH 8.0). In this study, we used the restriction enzymes BlnI, XhoI and SacI (Fermentas Life Sciences) as previously described by Arai et al., (2007) and Chen et al., (2008). Agarose-embedded DNA was digested for 4 h with 50 U of each endonuclease according to the manufacturer instructions. PFGE was performed on a CHEF DR III system (Bio-Rad) using 0.5 Tris-borateTable 1. Flavobacterium psychrophilum strains genotyped by PFGE. Strains Host species Origin (Chilean State) PFGE genotype with SacI XhoI BlnI 18250 Salmo salar Puerto Cisnes (XI) 4 2 2 19250 Salmo salar Puerto Cisnes (XI) 2 4 3 19443 Salmo salar Llanquihue (X) 5 1 2 1779 Salmo salar Rupanco (X) 3 5 1 1919 Salmo salar Quellón (X) 6 1 2 1150 Oncorhynchus mykiss Temuco (IX) 2 5 4 242 Oncorhynchus mykiss Curarrehue (IX) 2 6 3 1733 Oncorhynchus mykiss Antuco (VIII) 2 1 3 E6480 Oncorhynchus mykiss Melipeuco (IX) 2 5 4 1658 Oncorhynchus mykiss Ni 1 3 5 1731 Oncorhynchus mykiss Antuco (VIII) 3 5 1 1793 Oncorhynchus mykiss Ni 1 3 5 ATCC 49418T Oncorhynchus kisutch Washington 7 7 6 B97026 Oncorhynchus mykiss UK 7 7 6 ATCC: American Type Culture Collection; Ni: no information. Bull. Eur. Ass. Fish Pathol., 29(6) 2009, 187 EDTA buffer at 14oC and electrophoresis 18.5 h at a field angle 120o at 6 V cm-1 with pulse times of 1–12 s. DNA of Salmonella braenderup cleaved by XbaI was used as molecular size marker. Following electrophoresis, the gels were stained with ethidium bromide (0.5 μg ml–1) for 30 min, distained in distilled water for 1 h, and visualised with a UV transilluminator. All gels were also scanned and the images were captured by a Gel Doc-2000 gel documentation system (Bio-Rad). For the analysis and comparison of the PFGE patterns, a data analysis was performed by using the Diversity Database software (BioRad). The computed similarities among the isolates were estimated by means of the Dice coefficient (Sd) (Dice, 1945) as a measure of similarity, according to the equation: Sd [2A/ (2A + B + C)] · 100, where A is the number of matching bands and B and C are the numbers of bands present in one strain but not in the other. Dendrograms were produced on the basis of the unweighted average pair group method (UPGMA) using the Diversity Database software. The interpretation of PFGE patterns was performed using a criterion whereby isolates having ≥ 95% band similarity were considered identical clones. PFGE band similarity exceeding 80% was used as the criterion for cluster formation. When the SacI restriction enzyme was used, all rainbow trout isolates were clustered into group I except isolate 1731 and all Atlantic salmon isolates except 19250 clustered into group II. A similarity of 87.5% was found between the two clusters (rainbow trout and salmon) (Figure 1A). These results using PFGE SacI demonstrated that the Chilean F. psychrophilum strains can be distinguished by host origin (trout or salmon), as also reported by Chen et al., (2008) for this bacterial species. These authors found in American F. psychrophilum higher diversity among Coho salmon isolates relative to rainbow trout isolates. However, the current study observed higher diversity among rainbow trout isolates relative to Atlantic salmon isolates (91.4% and 93.3%, respectively). On the basis of these similarity values, Chilean F. psychrophilum isolates belonging to each cluster were considered genetically closely related. These findings are consistent with the genetic homogeneity detected among the same Chilean isolates using REP-PCR (Sd values of 89.7%) and RAPD (Sd of 94.5%) procedures (Valdebenito & Avendaño-Herrera, 2009). These typing methods are fast and simple (Romalde, 2005), but relationship between the F. psychrophilum genetic profile and the fish host were not evident by either REP-PCR or RAPD. In contrast, restriction digest patterns resolved by endonucleases BlnI and XhoI did not differentiate the Chilean F. psychrophilum isolates as shown with SacI; trout isolates were clustered together with Atlantic salmon strains (Table 1). Although using these enzymes a remarkable existence of subgroups within the Chilean cluster could be differentiated, even when constituted by a single bacterium (Figure 1B and C); approximately the same Dice coefficient values within and between groups was also obtained. In fact, an average band similarity of 83.2 ± 1.3% was found between all Chilean F. psychrophilum isolates with both endonucleases. Similar results using the same restriction enzymes have been Bull. Eur. Ass. Fish Pathol., 29(6) 2009, 188 Figure 1. Dendrogram of F. psychrophilum strains constructed using UPGMA, based on PFGE band patterns obtained using SacI (A), XhoI (B) and BlnI (C). PFGE band similarity exceeding 80% was used as the criterion for cluster formation. (Continued on p. 189). Figure 1B. Figure 1A. 1793 1658 1733 E6480