Isolation of Flavobacterium-like bacteria from diseased salmonids cultured in Chile

Bull. Eur. Ass. Fish Pathol., 28(5) 2008, 176 Isolation of Flavobacterium-like bacteria from diseased salmonids cultured in Chile P. Ilardi and R. Avendaño-Herrera* Laboratorio de Veterquímica, Camino Melipilla 5641, Cerrillos, Santiago, Chile. Abstract The biochemical, serological and molecular characteristics of 8-pigmented isolates phenotypically related to Flavobacterium psychrophilum were determined. The bacteria were isolated in 2006 from diseased Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss) cultured in the South of Chile. The F. psychrophilum type strain ATCC 49418T and isolate B97026, obtained from rainbow trout in UK, were included in the study as a comparison. Their potential to cause pathology in Atlantic salmon was also investigated. The 8 isolates were biochemically identical, however were completely different to F. psychrophilum. Slide agglutination and Dot blot allowed us to confirm that the isolates constituted a homogeneous, but distinctive serological group to the F. psychrophilum isolates. Similar homogeneity was observed using RAPD-PCR with identical DNA patterns obtained, regardless of the source of isolation or geographic origin. Therefore, any of the above tests can be used to discriminate these pigmented bacteria from F. psychrophilum. Virulence studies suggest that these isolates could be considered as a potential pathogen for salmonids, mainly in mixed cultures with F. psychrophilum. These fish showed typical signs of flavobacteriosis. Recent studies on the sequencing of the 16S rRNA gene, together with the phenotypic and biochemical properties obtained in this study permitted us classify these 8 isolates in the genus Chryseobacterium. Further molecular studies are in progress in order to know the species of these pigmented bacteria and determine the real risk for the salmonid culture. Introduction Flavobacterium psychrophilum is the causative agent of bacterial cold-water disease (BCWD) and rainbow trout fry syndrome (RTFS), an important disease that is thought to affect all haemorrhaging in exposed dermal tissue, and sometimes necrosis on the gills and eyes (see reviews Dalsgaard, 1993; Nematollahi et al., 2003). species of salmonid fish worldwide (see review Nematollahi et al., 2003). However, Detection of F. psychrophilum can be performed using molecular-based methods like less severe losses associated with this disease have also been reported in several non- polymerase chain reaction (PCR) (Bader & Shotts, 1998; Urdaci et al., 1998; Izumi et al., salmonid fish (Lehmann et al., 1991; Iida & Mizokami, 1996). The clinical signs in affected 2000; Wiklund et al., 2000), although definitive diagnosis must be supported by the isolation fish are characteristic gross lesions on the body surface in the form of ulcers, skin and of the bacterium on agar which characteristic yellow-pigmented colonies are produced. We muscle lesions on the flank or in peduncle area, loss of the epithelial surface and have recently isolated other yellowpigmented bacteria associated with diseases * Corresponding author’s E-mail: reavendano@yahoo.com Bull. Eur. Ass. Fish Pathol., 28(5) 2008, 177 in the fingerling stages of Atlantic salmon salmon (n = 3) and rainbow trout (n = 5) in (Salmo salar) and rainbow trout (Oncorhynchus mykiss). These microorganisms were often different farms located in X Region of Chile, were used in the present study (Table 1). isolated from mixed cultures of F. psychrophilum, being all of them initially Flavobacterium psychrophilum type strain ATCC 49418 T (serotype Fp T) and isolate B97026 diagnosed as member of the F. psychrophilum species. The aims of the present study were obtained from rainbow trout in UK (Faruk et al. 2002) were used as a comparison. Other to examine the biochemical, serological and molecular characteristics of these bacteria isolate, VQ-Au1, recently obtained from the skin of infected and dead fish was chosen only compared with those of the F. psychrophilum type strain, as well as their pathogenic for the pathogenicity studies. The bacteria were grown on Anacker Ordal (AOA; 0.5% potential in freshwater-farmed fish. tryptone, 0.05% yeast extract, 0.02% beef extract, 0.02% sodium acetate, pH 7.2) Material and methods Biochemical characterization of the isolates supplemented with 1.5% agar and incubated aerobically at 15ºC for 3–5 days. Stock cultures Eight isolates obtained in pure and mixed cultures from external ulcers and lesions, from were maintained frozen at – 80°C in Criobilles tubes (AES Laboratory). severe outbreaks of mortalities in Atlantic Flavobacterium psychrophilum strains Isolates present study Characteristics Host Cells form (n = 3) a (n = 5) b ATCC 49418T B97026 Salmo salar Oncorhynchus mykiss Oncorhynchus kisutch Oncorhynchus mykiss Osorno (Osorno) Chapo lake (Llanquihue) USA UK Rods Rods Long filamentous rods Long filamentous rods Yellow Yellow Yellow Yellow Gram - - - - Catalase + + + + Oxidase + + + + O/F - - - - Colony colour Congo red adsorption - - - - Flexirubin pigments + + + + Citrate Simmons - - - - Vogues-Proskauer - - - - Arginine decarboxylase - - - - Lisine decarboxylase - - - - Ornithine decarboxylase - - - - Table 1 continued. Bull. Eur. Ass. Fish Pathol., 28(5) 2008, 178 Flavobacterium psychrophilum strains Isolates present study Characteristics Host (n = 3) a (n = 5) b ATCC 49418T B97026 Salmo salar Oncorhynchus mykiss Oncorhynchus kisutch Oncorhynchus mykiss Osorno (Osorno) Chapo lake (Llanquihue) USA UK 0 - - - - Growth at (°C) 4 + + + + 15 + + + + 28 + + - - 37 - - - - 0 + + + + 1.0 + + + + 1.5 + + w w 3 + + - - 6 - - - - Growth with NaCl (%) Growth on Trypticase soy agar + + - - Nutrient agar + + + + R2A + + + + Marine agar 2216 - - - - Tryptone yeast extract agar + + + + Columbia blood agar + + - - - - - - DNase w w - - Urease - - - - ß-Galactosidase - - - - Aesculin + + - - Gelatine + + - + Starch - - - - Tween 80 - - w w MacConkey agar Enzyme activities Hydrolysis of a , VQ– 2206s; VQ–4836s and VQ–6316s , VQ–106r; VQ–5916r; VQ–5926r; VQ–5946r and VQ–5966r b Table 1. Differential phenotypic characteristic of Flavobacterium-like isolates from known F. psychrophilum strains included in this study. Number of isolates are show in parenthesis; +, Positive; –, Negative; w, weakly positive. Bull. Eur. Ass. Fish Pathol., 28(5) 2008, 179 using xazole (1.25/23.75), enrofloxacin (5), phenotypical properties, standard bacteriological tests (MacFaddin, 1984), and florfenicol (30) and oxytetracycline (30). The diameter of each zone inhibition was other biochemical tests (Bernardet et al., 2002; Avendaño-Herrera et al., 2004a). Biochemical determined after 48, and, if necessary, 72 h of incubation at 15°C. Reference strain Aeromonas reactions tested included: colony morphology and pigmentation, cell morphology, gliding salmonicida subsp. salmonicida ATCC 33658T was used as a control. motility, Gram-staining, cytochrome oxidase, catalase reaction (3% H 2 O 2 ), oxidation/ Identification of the isolates by PCR All isolates were identified fermentation reactions, Voges-Proskauer test, lysine descarboxylase, ornhitine descarboxylase, arginine descarboxylase, presence of cell wall-associated flexirubin type pigments and absorption of Congo red. Hydrolysis of the following substrates was determined using AOA as basal medium: gelatin, agar, starch, Tween 80, casein, DNA, blood and tyrosine. The growth temperature range was tested from 0 to 42°C on AOA. Growth in the presence of 0 to 10% (w/v) NaCl was also determined. Growth was detected on MacConkey agar, Simmons’ citrate agar, blood agar, R2A agar, Marine agar 2216, Nutrient agar, Trypticase Soy Agar and Triptone yeast extract agar. Further biochemical analysis were carried out using API 20E and API ZYM (bioMérieux) strips, according to the manufacturer’s instruction with the exception of the incubation temperature, which was fixed at 15°C. Antimicrobial tests were applied by disc diffusion method on Mueller-Hinton agar (MHA) with 1% NaCl, as well as the dilute versions of MHA medium, as recommended by the Clinical and Laboratory Standards Institute (CLSI 2006) for use with F. psychrophilum. The chemotherapeutic reagents used (Oxoid, (micrograms per disc)) were: amoxicillin (25), trimethoprim-sulfametho- Chromosomal DNA was extracted using Insta-Gene Matrix (Bio-Rad) for pure bacterial cultures according to the manufacturer ’s instructions. All PCR amplifications were done using the species-specific primer designed by Urdaci et al. (1998) and the commercial kit Ready-To-GoTM PCR beads (Amersham Pharmacia Biotech), which included all the reagents needed for the PCR reaction (buffer, nucleotides and Taq DNA polymerase), except the specific primers and DNA template. All amplifications were carried out in a PXE 0.5 Thermalcycler (Thermo Electron Corporation), and the products were electrophoresed in a 1.5% (w/ v) agarose gel visualized with ethidium bromide (Bio-Rad) and photographed under UV light. A 100-bp DNA ladder (Invitrogen) was used as a molecular mass marker. The presence of a single band of 1088 base pair (bp) was considered to be positive for identification of F. psychrophilum. Serological characterization Antisera against two representative isolates obtained from rainbow trout and Atlantic salmon, codified as VQ-5926r and VQ-6316s respectively, were prepared by intravenous injections of rabbits with formalin-killed cells (10 9 cells ml -1 ) according to Sørensen and Larsen (1986). To evaluate the antigenic crossreactivity among the Flavobacterium-like Bull. Eur. Ass. Fish Pathol., 28(5) 2008, 180 bacteria and F. psychrophilum species, all (Dice 1945). A dendrogram was produced on serological tests included polyclonal antiserum raised against ATCC 49418T and the basis of the unweighted average pair group method (UPGMA). strain B97026, which were also prepared in this study. Serological assays were carried out Virulence tests in fish according to Avendaño-Herrera et al. (2004a), using whole cell preparation and heat stable O-antigens of each isolate as well as the F. psychrophilum type strain ATCC 49418T, and B97026. The serological relationship between isolates was determined using agglutination (Toranzo et al., 1987), with strong and rapid agglutination considered positive. Dot blot analysis was also conducted as denoted by Cipriano et al. (1985). Controls were made with PBS and serum from non-immunised rabbits. Only a reaction similar to that exhibited by the homologous strain was scored as positive. Infectivity trials were conducted using healthy Atlantic salmon (average weight 8– 10 g) obtained from a hatchery with no history of flavobacteriosis located in the central of Chile. To make sure that they were free from F. psychrophilum and other freshwater pathogens, the fish samples (gills, mucus, skin and kidney) were subjected to standard microscopical and bacteriological examination, and also analysed by PCR (Urdaci et al., 1998). Fish were allocated at a rate of 15 fish per 10-L tank with freshwater, aerated and acclimatized for 72 h prior to bacterial challenge. Representative Flavobacterium-like RAPD-PCR analysis VQ-6316s and F. psychrophilum VQ-au1 were chosen for the trial. Virulence assays were The RAPD reactions were performed using Ready-To-Go TM RAPD analysis beads performed by immersion challenge according to Avendaño-Herrera et al. (2006). Three types (Amersham Pharmacia Biotech) following the protocol described by Avendaño-Herrera et of trials were conducted by direct inoculation with a bacterial suspension of 5 x 106 cells ml-1 al. (2004b). These commercial beads have been optimised for RAPD-PCR reactions and, as into sterile freshwater: a) isolate VQ-6316s; b) F. psychrophilum VQ-au1 and c) bacterial with the PCR beads above, contained buffer, nucleotides and Taq DNA polymerase; the mixtures of the both microorganisms (2.5 x 106 cells ml-1 of each strain). Fish without only reagents added to the reaction were template DNA (1 μl), 100 pmol of respective bacterial challenge were included as control. All trials were maintained in closed system RAPD primers (supplied in the kit) and water to make the reaction up to a volume of 25 μl. at 15 ± 1°C with a pH ranging from 7.6 to 7.8, and oxygen 7.8 mg L-1 using a 24L:0D light Amplification products were separated by horizontal electrophoresis and photographed regime. The fish were fed daily at 1.5% body weight and the water in each tank was under UV light. Data analysis on the resulting RAPD profiles was performed using changed once every-other-day to remove the faecal matter. GelCompar II software (Applied Maths). The computed similarities among strains were estimated by means of the Dice coefficient (Sd) Dead fish were removed from tanks daily for a 28 days period and subjected to microbiological analysis to confirm re-isolation of Bull. Eur. Ass. Fish Pathol., 28(5) 2008, 181 B Isolates RAPD pattern VQ-106r VQ-2206s VQ-4836s VQ-5916r VQ-5926r VQ-5946r VQ-5966s VQ-6316s 1 22.6 T ATCC 49418 B97026 20 40 60 80 2 100 % Similarity Figure 1. (A) Amplification fingerprints obtained for the isolates using RAPD with primer 5 (supplied with the kit). Lanes: MW, AmpliSize Molecular Ruler (100-bp DNA ladder, Invitrogen); 1 to 7, Flavobacterium-like pigmented bacteria and 8, Flavobacterium psychrophilum type strain. (B) Dendogram obtained using Dice similarity coefficient and unweighted pair group method average (UPGMA) analysis on the basis of RAPD profiles. Number along branches: % similarity. the inoculated strains. Once pure cultures of the pigmented isolates were obtained, were analysed and identified by slide agglutination MacConkey agar or Simmon’s citrate medium, while the F. psychrophilum strains did not grow assay and RAPD method as described above. on any of these media. The isolates did not produce acetoin (Voges–Proskauer test). Results and discussion Gelatin, aesculin and DNA were hydrolysed, but Tween 20, tyrosine, agar, starch and casein the isolates recovered from the diseased fish regardless of the host of isolation, and was very different to the F. psychrophilum isolates used as a comparison (Table 1). They were all Gram negative, rod-shaped, non-gliding and non-motile bacteria, catalase and cytochrome oxidase positive and were non fermentative. Colonies were smooth, shiny, circular with regular edges and yellow in colour. Growth occurred at 4–28°C and with 0–3% NaCl (this growth pattern is not typical for F. psychrophilum). All isolates contained a cell-wall-associated flexirubin-type pigment, but did not absorb Congo red. Growth on Columbia blood agar (a-haemolytic) and trypticase soy agar was observed, but not on were not. The results of the API 20E test were the same for all strains, including both F. Cumulative mortality (%) Biochemical homogeneity was seen among 100 80 60 40 20 0 1 4 7 10 13 16 19 22 25 28 Days Control VQ-6316s VQ-au1 VQ-6316s–VQ-au1 Figure 2. Percentage cumulative mortalities of Atlantic salmon challenged immersion with a bacterial suspension of 5 x 106 cells ml-1 into sterile freshwater of a) isolate VQ-6316s; b) F. psychrophilum VQ-au1 and c) bacterial mixtures of the both microorganisms (2.5 x 106 cells ml-1 of each strain). Fish without bacterial challenge were included as control. Bull. Eur. Ass. Fish Pathol., 28(5) 2008, 182 psychrophilum strains (code 0006024). It is The ATCC 49418T and Scottish isolates gave a important to point out that the API 20E rendered false positive for glucose clear product of the expected 1088 bp when PCR analysis was carried out, while products fermentation/oxidation when compared with results from traditional tube test. This of a lower molecular size were observed for all of the disease isolates (data not shown). difference might be due to the influence of the culture medium and/or initial inocula These results imply that the 8 isolates obtained from diseased fish do not belong to density (Ravelo et al., 2001). the species F. psychrophilum. On the other hand, results from the API ZYM test also showed complete homogeneity Serological characterization of the isolates by slide agglutination and Dot blot assays among all the disease isolates. Activity was not detected for melibiase, lactase, showed only a strong reaction with the antisera raised against rainbow trout and hyaluronidase, cellulose, alpha-mannosidase and alpha-fucosidase. As expected, for the F. Atlantic salmon isolates (VQ-5926r and VQ6316s), demonstrating that they belong to a psychrophilum strains, negative results were found for all the enzymes involved in same serological group (data not shown). However there was no cross–reaction with the carbohydrate metabolism (reactions 13 to 20 in API ZYM galleries). Therefore, the two F. antisera raised against the two F. psychrophilum isolates. The strains ATCC psychrophilum strains showed classical biochemical patterns for the bacterium, as 49418T and B97026 only reacted with their homologous antiserum, confirming that the detailed in the literature (Bernardet & Grimont, 1989; Lorenzen et al., 1997). pigmented isolates are antigenically distinct compared to F. psychrophilum. As expected no The antibiotic sensitivities of the isolates were the same, regardless of the media used, and reaction with the pre-immune serum was detected. 48 h was required before the results could be read. All isolates were resistant to PCR-based typing is an effective approach in the epidemiological study of diseases caused oxytetracycline and highly sensitive to the other drugs tested. This pattern of by various Gram-negative bacteria. In this study, RAPD analysis was applied to obtain antimicrobial sensitivity could possibly be explained by the common use of these drugs genetic fingerprints of the eight isolates, and to discriminate them from the F. psychrophilum in fish farms to control disease. In contrast, all F. psychrophilum strains were totally isolates. Initially RAPD analysis was performed using each of the six primers resistant to trimethoprim-sulfamethoxazole and highly susceptible to the remained provided in the kit. However, only one of the six primers, oligonucleotide P5, generated chemotherapeutic agents tested. Moreover, scant growth occurred in 48 h, but before 72 reproducible patterns with an appropriate number of amplified bands. The analysis of h clear and well-defined zones of inhibition were displayed in concordance with the the putative Flavobacterium isolates showed an identical profile among all strains with recommended by the CLSI (2006). amplification bands ranging from 500 to 2000 Bull. Eur. Ass. Fish Pathol., 28(5) 2008, 183 bp, indicating genetic homogeneity between to the fastidious nature of this microorganism, the isolates (Figure 1A). The type strain ATCC 49418T and Scottish isolate yielded a different as previously reported for F. psychrophilum (Michel et al., 1999). None of the control fish fingerprint ranging in size from 200 to 1300 bp, and were grouped within other cluster, died during the experiment. with a similarity level of 22.6% (Figure 1B). RAPD fingerprinting has previously been used to distinguish between F. psychrophilum and numerous other closely related bacteria found in diseased salmonid fish (Crump et al., 2001). Mechanisms of virulence and condition that influence virulence of the Flavobacterium-like species studied are unknown, while the pathogenicity of F. psychrophilum infections is not yet understood, and only scant data on virulence factor are available (Nematollahi et The results of the virulence testing with al., 2003). We speculate that the degree of pathogenicity or expression of the pathogenic Flavobacterium-like isolate (VQ-6316s) and VQ-au1 gave no mortalities and did not potential of disease-producing microorganisms is increased when both isolates are induce disease when fish were infected with the bacterium by bath. In contrast, prolonged mixed, since the isolates alone did not kill fish even when high concentrations were applied immersion (18h) of fish with a mixture of both isolates produced the first incidence of dead (data not shown). In fact, mixed infections of certain viral, bacterial, fungus or parasitic fish fish 11 days and cumulative mortalities of 100%, 23 days after the exposure to VQ-6316s– pathogens and F. psychrophilum are frequently observed in various salmonids fish species VQ-au1 (Figure 2). It is important to denote that the timing of immersion of fish (18 h) was (see review Cipriano & Holt, 2005). However, so far unknown whether the attachment, chosen based on our previous experience with challenge model for external pathogens colonization or subsequent protease and/or toxin released and penetration by the bacteria (Avendaño-Herrera et al., 2006), being the most similar to the natural route of infection. are triggered (Nematollahi et al., 2003). Possibly fish tissue is altered (in some way Gross external lesions were seen in all fish, typical of those seen in flavobacteriosis. by bacteria), which facilitates bacterial infection and further invasion. Microscopic examination of wet mount smears from the external lesions of dead fish revealed the presence of high quantities of long F. psychrophilum rods and shorter rods from Flavobacterium-like. A mixed culture of the two isolates was recovered from the lesions of only a few moribund fish. Based on the RAPD-PCR and confirmed by serological tests, all pure isolates obtained from the infected fish were identified as those used for the challenge. Failure to recover the bacterium on AOA from the other fish is probably due In summary, the pigmented bacteria isolated from the diseased rainbow trout and Atlantic salmon may be a potential pathogen for salmonid culture in Chile, mainly in mixed cultures with other fish pathogen such as F. psychrophilum. These isolates were biochemical, serological and genetically homogeneous, but were totally different to F. psychrophilum. Recent studies on the sequencing of the 16S rRNA gene, together with the phenotypic and biochemical Bull. Eur. Ass. Fish Pathol., 28(5) 2008, 184 properties obtained in this study permits us classify these 8 isolates in the genus Chryseobacterium. In this genus there are species known to be pathogen to fish (Mudarris et al., 1994; de Beer et al., 2006), as well as other isolates not yet identified at species level, but still causing mortalities to aquatic organisms (Bernardet et al., 2005). Further molecular studies are in progress in order to know the species of these pigmented bacteria and determine the real risk for the salmonid culture. Acknowledgement This work was supported by grant IPC 019 from the Program Bicentenario Ciencia y Tecnología, CONICYT– Chile. Thanks are expressed to the fish laboratories for sending the isolates. 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