Recovery of Edwardsiella piscicida from farmed whitefish, Coregonus lavaretus (L.), in Finland

Aquaculture 454 (2016) 19–26

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Recovery of Edwardsiella piscicida from farmed whitefish, Coregonus lavaretus (L.), in Finland Shafigh Shafiei a,1, Satu Viljamaa-Dirks b, Krister Sundell a, Sirpa Heinikainen b, Takele Abayneh c,2, Tom Wiklund a,⁎ a b c

Laboratory of Aquatic Pathobiology, Environmental and Marine Biology, Faculty of Science and Engineering, Åbo Akademi University, Turku, Finland Finnish Food Safety Authority Evira, Kuopio, Finland Department of Food Safety and Infection Biology, Section for Microbiology and Immunology, Norwegian School of Veterinary Science, Oslo, Norway

a r t i c l e

i n f o

Article history: Received 15 September 2015 Received in revised form 3 December 2015 Accepted 10 December 2015 Available online 11 December 2015 Keywords: Bacterial fish pathogen Coregonus Edwardsiella piscicida Whitefish

a b s t r a c t Bacterial pathogens, preliminary identified as Edwardsiella sp., were isolated from farmed whitefish, Coregonus lavaretus (L.), in Finland during disease outbreaks in 2000, 2002 and 2013. The diseased fish exhibited clinical and histopathological signs of general septicemia. Bacterial diagnostics were initially performed with conventional biochemical tests and the API50 CH rapid diagnostic system, followed by a species-specific PCR assay. Antimicrobial susceptibility profiles, enterobacterial repetitive intergenic consensus PCR (ERIC-PCR), multilocus sequence analysis (MLSA) and biofilm formation were used to characterize the bacterial isolates. For comparison, two reference strains Edwardsiella piscicida ET883T (NCIMB 14824) and Edwardsiella tarda ATCC 15947T were included in the analyses. Biochemical and antimicrobial analysis displayed the same profile for all isolates from whitefish and this was similar to both reference strains. Molecular assays including PCR and ERIC-PCR demonstrated that all whitefish isolates were genetically different from the E. tarda reference strain. MLSA analysis revealed that the whitefish isolates and ET883T are in a separate clade distinct from ATCC 15947T; they are not genetically identical. It is concluded that whitefish is susceptible to infection with E. piscicida a bacterial species with a wide geographical distribution and adaptability to different hosts. It is postulated that in the future E. piscicida might pose a serious threat to farmed whitefish especially in recirculating aquaculture systems. Statement of relevance: Edwardsiella piscicida was recently described and separated from the species Edwardsiella tarda. E. tarda/piscicida is a well-known fish pathogen infecting mainly warm water species. However, during recent years the pathogen has also been isolated from cold water fish species, mainly salmonids. It is not known if this is due to an adaptation of the pathogen to lower temperatures or due to farming of these fish in higher temperatures. In the present study E. piscicida was isolated and described for the first time from disease outbreaks of whitefish (salmonid species) in different freshwater farms in Finland. Farming of whitefish is increasing in Finland, especially in recirculating systems. The examined isolates were all obtained from farmed whitefish and it is hypothesized that this pathogen could be an increasing threat for the future farming of whitefish. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Edwardsiella tarda has previously been described as a pathogen of fish, reptiles, birds, amphibians, aquatic invertebrates and terrestrial mammals including humans (see review in Mohanty and Sahoo, ⁎ Corresponding author at: Laboratory of Aquatic Pathobiology, Environmental and Marine Biology, Faculty of Science and Engineering, Åbo Akademi University, BioCity, Artillerigatan 6, 20520 Turku, Finland. E-mail address: twiklund@abo.fi (T. Wiklund). 1 Present address: Department of Aquatic Animal Health, Faculty of Veterinary Medicine, University of Tehran, Tehran, Iran. 2 Present address: College of Veterinary Medicine and Agriculture, Addis Ababa University, Debre-zeit, Ethiopia.

http://dx.doi.org/10.1016/j.aquaculture.2015.12.011 0044-8486/© 2015 Elsevier B.V. All rights reserved.

2007). In fish, edwardsiellosis is a systemic disease with the potential to evoke mass mortality; this has occurred in more than 20 species of freshwater and marine fish, (Abbott and Janda, 2006; Mohanty and Sahoo, 2007). Edwardsiellosis occurs mainly in warm water, although occasional disease outbreaks have been reported in cold water species including chinook salmon, Oncorhynchus tshawytscha (Walbaum) (Amandi et al., 1982), rainbow trout, Oncorhynchus mykiss (Walbaum) (Reddacliff et al., 1996; Řehulka et al., 2012), brook trout, Salvelinus fontinalis (Mitchill) (Uhland et al., 2000), and turbot, Scophthalmus maximus (L.) (Nougayrede et al., 1994; Padrós et al., 2006). In Europe, in addition to their detection in rainbow trout (Reddacliff et al., 1996; Řehulka et al., 2012) and turbot (Castro et al., 2006; Nougayrede et al., 1994; Padrós et al., 2006), bacteria described as E. tarda have been

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S. Shafiei et al. / Aquaculture 454 (2016) 19–26

isolated from the European eel, Anguilla anguilla (L.) (Abayneh et al., 2012a; Alcaide et al., 2006). It has been demonstrated that E. tarda isolates from fish displayed phenotypic and genetic features distinct from other E. tarda isolates: this has been interpreted as evidence for the existence of unrecognized taxa within the genus Edwardsiella (Abayneh et al., 2012a; Acharya et al., 2007; He et al., 2011; Yang et al., 2012). The findings of previous studies resulted in the assignation of a new taxon within the genus Edwardsiella, Edwardsiella piscicida, which was described from fish in Europe and Asia (Abayneh et al., 2012b). A recent study also confirmed the presence of E. piscicida in diseased fish in the southeastern United States (Griffin et al., 2014). A bacterial pathogen preliminarily identified as an Edwardsiella sp. was isolated from three disease outbreaks (2000, 2002 and 2013) in farmed whitefish, Coregonus lavaretus (L.) in freshwater in Finland. The aim of the present study was to identify and characterize the bacterial isolates obtained from these disease outbreaks in whitefish. 2. Materials and methods 2.1. Fish In August 2000, increased mortality was recorded in whitefish (25 g) reared in net cages in a small lake in eastern Finland (Farm 1), with a high water temperature (20–23 °C) and decreased water flow. The mortality was high, reaching 40% in some fish populations. During late summer 2002 (water temperature 20 °C), another episode of mass mortality occurred in whitefish (50 g) in the same fish farm. A third epizootic was reported in whitefish (400–1000 g) in May 2013 from a recirculation freshwater fish farm (Farm 2) with water temperatures around 17–20 °C. Moribund whitefish from the different disease outbreaks were analyzed for the presence of bacterial pathogens in their internal organs. 2.2. Laboratory examination of whitefish The whitefish from 2000 (n = 20) and 2002 (n = 11) were studied pathologically and bacteriologically according to routine diagnostic procedures (Midtlyng et al., 2000). Farmed fish in the area were being monitored for viral diseases and were considered at the time to be free of all primary viral fish diseases and therefore no further virological examination was conducted. Samples of the internal organs (spleen, kidney, heart, liver and pyloric ceca) of whitefish from 2000 (n = 4) and 2002 (n = 3), were fixed in 10% neutral buffered formalin and prepared for histopathological examination according to routine methods. The whitefish from 2013 (n = 10) were studied only macroscopically and bacteriologically. Samples of kidney and/or spleen of whitefish were inoculated onto blood agar containing 5% bovine blood or tryptic soy agar (TSA, Becton, Dickinson & Company, BD, Sparks, MD, USA) and incubated for 10 days at 15 or 20 °C. 2.3. Bacterial isolates Four bacterial isolates from the 2000 and 2002 disease outbreaks and seven isolates from the disease outbreak in 2013 were subcultivated and included in the present study (Table 1). Two reference strains, E. piscicida (NCIMB 14824, ET883T), and E. tarda (ATCC 15947T) were also included for comparison. Stock suspensions of all isolates were kept in tryptic soy broth (TSB, Difco) with 20% glycerol at −80 °C. Bacterial isolates were cultured at 25 °C on TSA. Flavobacterium psychrophilum (P7-1A/10; Sundell et al., 2013) was used in the biofilm assay as a positive control.

Table 1 Bacterial isolates used in the present study. Isolate

Host

Origin

Isolation year

P6-2B/13 P6-3B/13 P6-5/13 P6-6/13 P6-7/13 P6-8/13 P6-10/13 1986/00 2558/02/1 2558/02/2 2558/02/3 ET883T (NCIMB 14824) ATCC 15947T

Whitefish Whitefish Whitefish Whitefish Whitefish Whitefish Whitefish Whitefish Whitefish Whitefish Whitefish European eel Human feces

Finland Finland Finland Finland Finland Finland Finland Finland Finland Finland Finland Norway Sweden

2013 2013 2013 2013 2013 2013 2013 2000 2002 2002 2002 1989 1959

Droppers), oxidative-fermentative metabolism of glucose (MacFaddin, 1980), H2S production (sulfide indole motility medium SIM, BD). Cell motility was determined by microscopy of broth culture and inoculation of SIM. The morphology of the bacteria was studied by Gram staining (Collins et al., 2004). The test media were read after incubation for 24 h at 25 °C. In addition, an API 50 CH (bioMérieux, Marcy I'Etoile, France) rapid diagnostic kit was used to determine the fermentation of 49 carbohydrates according to the manufacturer's instructions. The strips were incubated at 25 °C for 48 h. Growth at different temperatures was evaluated by inoculating bacterial isolates in TSB and incubating at 12, 40 and 42 °C for 24 h. The turbidity of the inoculated media was evaluated by the naked eye. 2.5. PCR assay Identification of all isolates was done with a PCR specific for E. piscicida and E. tarda. The DNA template was obtained by suspending a colony from a 48 h TSA culture in 50 μL double-distilled water (ddH2O). One microliter of the suspension was used as the template in a PCR reaction including 1 × KAPA2G™ Robust HotStart Ready Mix, 0.5 μM of forward and reverse primers specific for E. tarda (5′-CAG TGA TAA AAA GGG GTG GA-3′ and 5′-CTA CAC AGC AAC GAC AAC G-3′), or E. piscicida (5′-CTT TGA TCA TGG TTG CGG AA-3′ and 5′-CGG CGT TTT CTT TTC TCG-3′) (Griffin et al., 2014) and ddH2O to a total volume of 25 μL. The thermal conditions for the amplification reaction were as follows: initial denaturation for 2 min at 95 °C followed by 34 amplification cycles including denaturation for 15 s at 95 °C, annealing of primers for 15 s at 60 °C, and extension for 15 s at 72 °C, with a final extension step for 5 min at 72 °C. The amplified PCR products were electrophoresed (4 V cm−1, 30 min) on a 1% agarose gel stained with ethidium bromide and visualized under UV light. 2.6. Enterobacterial repetitive intergenic consensus -PCR Enterobacterial repetitive intergenic consensus (ERIC)-PCR was used to determine the genetic relationship between the isolates from whitefish and the two reference strains (ET883T, ATCC 15947T). Genomic DNA of the isolates was extracted using Instagene TM Matrix (Bio-Rad) according to the manufacturer's instructions and amplified with the primer ERIC1R (Versalovic et al., 1991). The PCR was repeated twice and the similarity of the profiles was analyzed by taking into consideration the stability of the bands. The profiles were compared with BioNumerics software using an unweighted pair group method with arithmetic mean (UPGMA) and the Dice coefficient. 2.7. Multi-locus sequence analysis

2.4. Characterization of the isolates The biochemical characterization of the bacterial isolates included catalase production, cytochrome oxidase (BD™ Oxidase Reagent

Multi-locus sequence analysis (MLSA) of isolates from the 2013 outbreak was carried out using the protocol described by Abayneh et al. (2012a). Housekeeping gene sequence data of E. piscicida isolates from

S. Shafiei et al. / Aquaculture 454 (2016) 19–26

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Table 2 Nucleotide accession numbers of Edwardsiella isolates used in MLSA. Edwardsiella isolate

ET883 ET2640 ET2639 ET2455 ET3381 ET3612 ET2493 LTB4 ETA1 ETB1 NCIMB 2056 7150HSF6 M07.1 AL93 379 ATCC 15947 NCIMB 2034 9314 9315 9316 9317 9318 9319 9320

Species

E. E. E. E. E. E. E. E. E. E. E. E. E. E. E. E. E. E. E. E. E. E. E. E.

piscicida piscicida piscicida piscicida piscicida piscicida piscicida piscicida piscicida piscicida piscicida ictaluri ictaluri ictaluri ictaluri tarda tarda piscicida piscicida piscicida piscicida piscicida piscicida piscicida

Origin

Norway Norway Norway Norway Norway Norway Norway China UK UK USA USA USA USA ATCC collection NCIMB collection Finland Finland Finland Finland Finland Finland Finland

Genes, sequence size used and accession numbers

Reference

gyrB (…bp)

Mdh (…bp)

adk (…bp)

dnaK (…bp)

phoR (…bp)

metG (…bp)

aroE2 (…bp)

JN700525 JN700729 JN700728 JN700726 JN700730 JN700731 JN700727 JN700740 JN700732 JN700535 JN700741 JN700744 JN700746 JN700745 JN700747 EU259314

JN700555 JN700558 JN700557 JN700554 JN700559 JN700560 JN700556 JN700572 JN700561 JN700564 JN700576 JN700581 JN700580 JN700579 JN700578 JN700577 JN700575

JN700687 JN700644 JN700643 JN700641 JN700645 JN700646 JN700642 JN700657 JN700648 JN700651 JN700659 JN700664 JN700662 JN700663 JN700665 JN700641 JN700658

JN700529 JN700533 JN700532 JN700530 JN700534 JN700535 JN700531 JN700547 JN700536 JN700540 JN700553 JN700552 JN700550 JN700549 JN700551 JN700539 JN700543

JN700669 JN700673 JN700672 JN700670 JN700674 JN700675 JN700671 JN700687 JN700676 JN700680 JN700688 JN700693 JN700691 JN700689 JN700692 JN700679 JN700683

JN700585 JN700589 JN700588 JN700586 JN700590 JN700591 JN700587 JN700603 JN700592 JN700596 JN700604 JN700609 JN700607 JN700606 JN700608 JN700595 JN700599

JN700613 JN700617 JN700616 JN700614 JN700618 JN700619 JN700615 JN700629 JN700620 JN700623 JN700630 JN700637 JN700635 JN700634 JN700636 JN700633 JN700632

Abayneh et al. (2012a) ” ” ” ” ” ” ” ” ” ” ” ” ” ” ” ” This study This study This study This study This study This study This study

Norway, China, UK and NCIMB collection as well as isolates of Edwardsiella ictaluri and reference E. tarda strains were also used along with Finnish isolates from whitefish in the current MLSA (Table 2). Housekeeping gene sequence data from whole genome sequence of E. tarda strain ATCC 23685 (acc. no. NZ-ADGK01000012), E. tarda strain EIB202 (acc. no. CP001135.1) and E. ictaluri strain 93–146 (acc. no. CP001,600.1) were also included in the MLSA. Seven gene loci previously used in MLSA were selected and amplified; these included adk (adenylate kinase), dnaK (molecular chaperone), gyrB (DNA gyrase subunit B), phoR (phosphate regulon sensor protein), metG (methionyl-tRNA synthetase), mdh (malate/lactate dehydrogenase) and aroE2 (shikimate 5-dehydrogenase). Genomic DNA was extracted by the DNeasy blood and tissue kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. PCR amplification of each gene was performed in a 50 μL reaction volume using 5 μL of 10 × key buffer, 4 μL of MgCl2, 1 μL of deoxynucleoside triphosphate mix, 1 μL of each primer, 0.4 μL of Taq polymerase, 33.6 μL of sterile distilled water and 4 μL of template DNA. The PCR amplification conditions were initial denaturation at 94 °C for 3 min, 35 cycles of denaturation at 94 °C for 45 s, annealing at 55 °C for 45 s and extension at 72 °C for 2 min followed by final extension at 72 °C for 5 min. Amplified products were purified with the QIAquick PCR purification kit and sequenced commercially (GATC Biotech, Konstanz, Germany). Evolutionary analyses of concatenated sequences were conducted in the molecular evolutionary genetics analysis (MEGA) software package, version 5. Concatenation of the housekeeping gene sequences was done in a head-to-tail manner in the order of their physical position in the whole genome as described in Abayneh et al. (2012a). Phylogenetic trees were constructed by neighbor-joining (NJ), evolutionary distance was calculated using the Kimura two-parameter model and statistical support for the resulting node was performed by the bootstrap method, based on 1000 replications.

Diagnostics), tetracycline (30 μg, Oxoid), oxolinic acid (2 μg, Oxoid) and trimethoprim/sulfamethoxazole (1.25/23.75 μg, Oxoid). Pure cultures of the bacterial isolates were suspended in 5 mL sterile 0.5% NaCl to a density of 1–2 on the McFarland's nephelometer standards. Then, 20 μL of the bacterial suspensions were diluted in 5 mL 0.5% NaCl and spread onto the Mueller Hinton agar plates. Antibiotic disks were placed onto the agar plates after any excess suspension had been removed and the agar plate had been allowed to dry. The zone of bacterial growth inhibition was measured after 48 h of incubation at 25 °C. Results were considered as sensitive, intermediate and resistant according to the standard measurement of inhibition zones in mm (Casals and Pringler, 1991). The Minimum Inhibitory Concentrations (MICs) of flumequine (FLU), oxytetracycline dihydrate (OTC) and trimethoprim (TMP) (Sigma) were determined for the bacterial isolates using a broth microdilution method according to guidelines from the Clinical and Laboratory Standards Institute (CLSI, 2005) with some modifications. In these tests, the antimicrobial agents were dissolved in appropriate solvents including methanol, 0.03 M NaOH and dimethyl sulfoxide (for OTC, FLU and TMP, respectively) to make stock solutions and then further two fold diluted in TSB such that the concentration of all the antibiotics ranged from 0.006 to 50 μg mL−1. The optical density (OD) of broth cultures was adjusted to 0.450 ± 0.005 at 520 nm (Unicam, Heλios β), corresponding to an approximate bacterial density of 109 CFU mL−1 and subsequently diluted 1:1000 in TSB. One hundred microliters of the final suspension was inoculated in triplicate into wells of U-bottomed 96-well polystyrene microtitre plates (Nunc) containing 100 μL of two fold serial dilutions of the tested antimicrobials. The microplates were incubated for 48 h at 25 °C and subsequently the bacterial growth in the wells was observed visually against a dark background. The MIC values of the tested antibiotics against bacterial isolates were interpreted as the lowest antimicrobial concentration inhibiting visible growth after 48 h incubation.

2.8. Antimicrobial susceptibility test

2.9. Biofilm assay

The susceptibility of the bacterial isolates to different antibiotics was analyzed with the disk diffusion method on Mueller Hinton agar (Difco, BD, USA). The antibiotics tested included florfenicol (30 μg, Mast

The biofilm forming ability of the different isolates was examined according to the protocol described by Xiao et al. (2009) with some modifications. The bacterial cells were suspended in TSB and the optical

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S. Shafiei et al. / Aquaculture 454 (2016) 19–26

whitefish from all three disease outbreaks. Eleven isolates were purified and selected for further study (Table 1). All tested isolates were Gram-negative, motile short rods, catalase positive, oxidase negative, H2S positive, with facultative anaerobic metabolism of glucose. All studied isolates were capable of growth at 12, 40 and 42 °C, except for the E. tarda reference strain (ATCC 15947T) that grew only weakly at 12 °C. The API 50 CH test showed the same profile for all isolates and this was similar to the reference strains in that they were able to ferment D -ribose, D -galactose, D -glucose, D -fructose, D -mannose, N-acetylglucosamine, D -maltose, L -fucose and potassium gluconate (Table 3).

Table 3 Results of carbohydrate fermentation (API-50 kit) of bacterial isolates from whitefish and Edwardsiella reference strains, after 48 h incubation at 25 °C. 1–11, whitefish isolates; 12, E. piscicida reference strain (ET883T, NCIMB 14824); 13, E. tarda reference strain (ATCC 15947T); +, positive reaction; −, negative reaction. Fig. 1. Focal to coalescing necrotic foci in the peritoneum and accumulation of inflammatory cells in the pancreas of one-summer old whitefish infected by E. piscicida. Black arrow: pyloric cecum. White arrow: intact exocrine pancreatic cells. White stars: lymphocytic accumulation. Black stars: necrotic cells.

densities were adjusted to 0.5 at 600 nm. The cell suspensions were diluted 1:20 in TSB and 200 μL of the diluted suspensions were inoculated in triplicate into the wells of a 96-well flat-bottomed microtitre plate (Nunc). In addition, wells containing TSB or F. psychrophilum cells (using TYES broth) were used as negative and positive controls, respectively. After 3 days of incubation at 25 °C, media and unattached bacterial cells were removed and the wells were gently washed with tap water (2×). The attached cells were stained with 0.1% crystal violet (Merck) for 45 min at room temperature after which the wells were rinsed (2 ×) with tap water. Finally, the bound dye was extracted from the stained cells by using 200 μL 96% ethanol and the plates were placed on a shaker (200 rpm, 15 min) to achieve full release of the dye. Biofilm formation was quantified by measuring the absorbance of the solution at 595 nm (plate reader, Victor2, PerkinElmer Wallac, Finland).

Characteristic

12

13

D-Arabinose

− − −

− − −

− − −

L-Arabinose

−

−

−

D-Ribose

+

+

+

D-Xylose

−

−

−

L-Xylose

−

−

−

D-Adonitol Methyl-ßD-xylopyranoside

−

−

−

D-Galactose

− +

− +

− +

D-Glucose

+

+

+

D-Fructose

+

+

+

D-Mannose

+

+

+

L-Sorbose

−

−

−

L-Rhamnose

−

−

−

Dulcitol

−

−

−

Inositol

− −

− −

− −

−

−

−

D-Celiobiose

− − + − − − − −

− − + − − − − −

− − + − − − − −

D-Maltose

+

+

+

−

−

−

D-Melibiose

−

−

−

D-Saccharose

−

−

−

D-Trehalose

−

−

−

Inulin

− −

− −

− −

−

−

−

D-Turanose

− − − − −

− − − − −

− − − − −

D-Lyxose

−

−

−

D-Tagatose

−

−

−

D-Fucose

−

−

−

L-Fucose

+

+

+

Glycerol Erythritol

D-Mannitol

3. Results 3.1. Gross pathology and histopathology The infected whitefish showed signs of generalized septicemia, including exophthalmia, hemorrhagic congestion on the skin and at the base of fins, swelling and hyperemia of the anal region, hemorrhages in the internal organs, gills and muscle tissue, mottled liver, splenomegaly and renomegaly. Histological examination of the younger fish from the 2000 outbreak revealed the presence of focal necrosis in the peritoneal cavity, mainly affecting the pyloric area (Fig. 1). In some cases, generalized peritonitis was detected with inflammatory cells on most of the visceral organs. Gram-negative rod-shaped bacteria were identified in the peritoneal cavity in the affected areas. However, exocrine pancreatic cells were not affected to any major extent, and the pronounced influx of lymphocytes lining the pancreatic cells was more likely due to some nutritional imbalance. Necrotic foci were also seen in the kidney and single cell necrosis and small necrotic foci were detected in the liver. In fish from the 2002 outbreak, the histopathological changes were mostly limited to the kidney, with necrotic areas observed in the interstitial tissue and the tubular epithelium. There were also signs of necrosis in the liver and occasional bacterial emboli in the veins of the liver and the heart. 3.2. Phenotypic characterization of the bacterial isolates Pure cultures of white, cream-colored colonies were obtained from the kidney and occasionally from spleen or brain of the examined

Isolate number 1–11

D-Sorbito Methyl-αD-mannopyranoside Methyl-αD-glucopyranoside N-Acetylglucosamine Amygdalin Arbutin Esculin ferric citrate Salicin

D-Lactose

(bovine origin)

D-Melezitose D-Raffinose Amidon (starch) Glycogen Xylitol Gentiobiose

D-Arabitol

−

−

−

L-Arabitol

−

−

−

Potassium gluconate Potassium 2-ketogluconate Potassium 5-ketogluconate

+ − −

+ − −

+ − −

S. Shafiei et al. / Aquaculture 454 (2016) 19–26

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3.3. PCR and ERIC-PCR

3.6. Biofilm formation

All whitefish isolates and the E. piscicida reference strain (ET883 T ) were PCR positive using primers specific for E. piscicida, while no amplification was observed for the E. tarda reference strain (ATCC 15947T). In contrast, no positive amplification was detected from whitefish isolates or the E. piscicida reference strain (ET883 T ), although robust amplification was observed from the E. tarda reference strain (ATCC 15947T) using primers specific for E. tarda. In the ERIC-PCR, the whitefish isolates from 2000 and 2002 had only minor differences in the weaker bands (Fig. 2). Between the isolates from these two cases and the third whitefish epizootic, similar profiles with some variation were obtained. Slight variations were also seen between the isolates from the same epizootic. The reference strain ET883T differed from the whitefish isolates in that it displayed a strong band at about 1600 Kb that was much less pronounced in the whitefish isolates. There were also differences with respect to the weaker bands. All fish isolates differed clearly from the E. tarda reference strain (ATCC 15947T).

No clear difference in biofilm formation was observed among the whitefish isolates (Fig. 4). The E. tarda reference strain (ATCC 15947T) differed from the other isolates by displaying a two-fold increase in the OD value compared to the isolates from whitefish and the E. piscicida reference strain. On the other hand, biofilm formation of all whitefish isolates was significantly less extensive than that encountered with the F. psychrophilum positive control.

3.4. MLSA A phylogenetic tree constructed from concatenated sequence alignments of seven gene loci showed five genotypic clusters (Fig. 3). All E. piscicida isolates from different geographical regions, NCIMB2056 strain and whitefish isolates were resolved into a separate clade, while the NCIMB 2034 strain clustered with the E. tarda reference strain (ATCC 15947T). The E. tarda reference strain (ATCC 23685) comprised a separate clade. The E. ictaluri isolates formed a cluster more closely related to E. piscicida isolates than the E. tarda reference strain. 3.5. Antimicrobial susceptibility testing All 11 isolates and the two reference strains were similar in their sensitivity to the antibiotics used in this study; the isolates were susceptible to florfenicol (inhibition zone diameter [IZD] ≥ 25 mm), tetracycline (IZD ≥ 23 mm), oxolinic acid (IZD ≥ 32 mm) and trimethoprim + sulfamethoxazole (IZD ≥ 28 mm). The obtained MIC values of tested antimicrobials against the bacterial isolates are presented in Table 4. The MIC values of flumequine were between 0.05 and 0.1 μg mL−1. The oxytetracycline dihydrate MIC values were between 0.2 and 0.78 μg mL−1. All isolates had the same MIC values for trimethoprim (0.05 μg mL−1) except for the E. tarda reference strain (0.1 μg mL−1).

4. Discussion Although edwardsiellosis is generally described as a bacterial disease of warm water fish, occasional Edwardsiella infections in salmonids have been reported previously (Amandi et al., 1982; Reddacliff et al., 1996; Řehulka et al., 2012; Uhland et al., 2000). The present study investigating disease outbreaks in whitefish supports the conclusion that E. piscicida can affect also cold water fish species, like salmonids. Isolation of E. piscicida from whitefish has not been described previously. These findings suggest that any future increase in the farming of whitefish in Finland and possibly in other countries like Norway (Siikavuopio et al., 2013) and Russia, might be followed by a growing number of disease outbreaks attributable to E. piscicida. Indeed, in summer 2015, a fourth septicemic disease outbreak caused by E. piscicida was detected in whitefish in a Finnish farm using recirculating water (own unpublished results). While recirculation aquaculture systems (RAS) have become more popular due to their lower environmental impact compared to flow through systems, specific disease problems may arise demanding scrupulous attention to biosecurity and disease management. A high water temperature has been thought to be a predisposing factor for the disease outbreaks caused by E. tarda/piscicida, as earlier reported (Uhland et al., 2000; Zheng et al., 2004). However, the disease outbreak in 2013 in a recirculation system with temperatures below 20 °C as well as a report of an outbreak in rainbow trout at 12 °C (Řehulka et al., 2012) show that E. tarda/piscicida infections in fish do not only occur at higher water temperatures. Any stressful situation seems to be able to trigger E. tarda/piscicida induced mortality in salmonid fish. The clinical symptoms, macroscopic lesions and histopathological findings of whitefish in this study were not pathognomonic for edwardsiellosis, i.e. these pathological changes are similar to many other bacterial systemic infections. Abscess formation was not seen in whitefish, although this has been observed in many other fish species (Miyazaki and Kaige, 1985; Padrós et al., 2006) including rainbow trout (Reddacliff et al., 1996) infected with E. tarda/piscicida. It is possible that

Fig. 2. PCR amplification of enterobacterial genomic DNA by ERIC oligonucleotide primer ERIC1R and the similarity of the profiles (UPGMA). P6-5/13, P6-6/13, P6-2B/13, P6-3B/13, P6-10/13, P6-7/14, P6-8/13: isolates from Farm 2. 2558/02/1, 2558/02/2, 2558/02/3, 1986/00: isolates from Farm 1, ET883T (NCIMB 14824): Edwardsiella piscicida. ATCC15947T: Edwardsiella tarda.

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Fig. 3. Neighbor-joining phylogenic tree inferred from concatenated sequences of seven housekeeping genes obtained from 27 Edwardsiella isolates. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree.

the necrotic foci seen in several organs in this study, with time could have evolved into abscesses. Based on the biochemical and biophysical reactions obtained in the present study, all 11 isolates from whitefish showed phenotypic homogeneity to the E. piscicida reference strain. However, the whitefish isolates were also almost indistinguishable from the E. tarda reference

strain with respect to the phenotypic results, except for their better growth capacity at 12 °C, evidence of the close phenotypic similarities between these two species. This indicates that routine bacteriological techniques and metabolic profiling are not sufficient if one wishes to make a proper differentiation of the two Edwardsiella species

Table 4 Distribution of the Minimal Inhibitory Concentration (MIC) of different antimicrobial agents against the examined Edwardsiella sp. isolates. Antimicrobial agent

Bacterial isolate

Number of bacterial isolates displaying MIC (μg mL−1) ≤0.025 0.05 0.10 0.2 0.39 0.78 1.56

Flumequine

Oxytetracycline dihydrate

Trimethoprim

T

ATCC 15947 ET883T Whitefish isolates ATCC 15947T ET883T Whitefish isolates ATCC 15947T ET883T Whitefish isolates

1 1 8

3 1 1 6

1 1 11

4

1 Fig. 4. Biofilm formations expressed as an average of three replicates of the optical density (595 nm) of stained cells of isolates from whitefish and reference strains. 1, P6-2B/13; 2, P6-3B/13; 3, P6-5/13; 4, P6-6/13; 5, P6-7/13; 6, P6-8/13; 7, P6-10/13; 8, 1986/00; 9, 2558/02/1; 10, 2558/02/2; 11, 2558/02/3; 12, ET883T (NCIMB 14824); 13, ATCC 15947T; control: TSB; F. psychrophilum: positive control.

S. Shafiei et al. / Aquaculture 454 (2016) 19–26

(Abayneh et al., 2012b; Griffin et al., 2013). Despite the close phenotypic relationship, the molecular assays including PCR and ERIC-PCR demonstrated that all whitefish isolates were clearly different from the E. tarda reference strain. In addition, the phylogenetic analysis based on MLSA of seven housekeeping genes revealed that all whitefish isolates were distinct from the E. tarda reference strains (ATCC 15947T, ATCC 23685). These results support previous studies concerning genetic divergence between bacterial isolates from fish and isolates from humans (Abayneh et al., 2012a,b; Griffin et al., 2013, 2014; Yang et al., 2012). The high evolutionary divergence of the cluster containing reference isolates (ATCC 15947T, ATCC 23685 and NCIMB 2034) compared with the other clusters in the MLSA indicates that E. tarda infecting humans may have evolved differently from the E. piscicida strains infecting fish. This may be due to specific host or niche adaptation and it also highlights the limited role of human isolates in the epidemiology of fish edwardsiellosis and vice versa. The occurrence of an E. piscicida isolate clade closer to E. ictaluri than to the E. tarda reference isolate cluster, points to some genetic relationship between E. piscicida and E. ictaluri. The uniformity of the ERIC-PCR profiles of the E. piscicida isolates from the same farm in 2000 and in 2002 (Farm 1), suggests that they have a common origin. However, the differences between these isolates and the isolates from the third disease outbreak in Farm 2 in 2013 do not support the concept of a common origin. The relative similarity of the Finnish isolates to the reference strain (ET 883T) originating from a different fish species (European eel, A. anguilla) and a different country (Norway; Abayneh et al., 2012a) shows that ERIC-PCR might not be the best method for tracing the epidemiologic links of edwardsiellosis with respect to geographical origin and fish host. Another, although less likely explanation of the similarity, would be that all the fish isolates share a common origin and that the pathogen has already a wide geographic distribution. The use of multilocus variable number tandem repeat analysis (MLVA) capable of discriminating between E. piscicida isolates with respect to host and outbreak sources (Abayneh et al., 2013), could be an appropriate method to determine the epidemiological connections of E. piscicida isolates of different geographical, host or outbreak sources. A possible source of E. piscicida could be the highly migratory European eel, since E. tarda/piscicida has been isolated from diseased eels in Spain (Alcaide et al., 2006), Norway (Abayneh et al., 2012a) and Sweden (E. Jansson, pers. Comm., unpublished results). The similar sensitivity patterns of all whitefish isolates as well as the reference strains to the tested antibiotics is in agreement with previous reports of the antimicrobial profiles for Edwardsiella species (Alcaide et al., 2006; Stock and Wiedemann, 2001). However, in view of the likely expansion of whitefish farming, especially in RAS, the incidence of edwardsiellosis may increase, necessitating the recurrent use of antibiotics, which may create favorable circumstances for the development of resistant strains in the future. The formation of a biofilm is considered to be a major factor in the pathogenesis, resistance and spread of bacteria (Costerton et al., 1999; Sundell and Wiklund, 2011). When the biofilm-forming ability of the whitefish isolates was examined, it was found that the isolates were not capable of forming thick biofilms and thus differed from the positive control (F. psychrophilum). These results are in agreement with a previous study which investigated the biofilm forming capacity of E. tarda isolates from different fish species (He et al., 2011). However, the weak biofilm forming capacity of the examined whitefish isolates suggests that this trait is not very important for the survival of this pathogen in this environment. In conclusion, E. piscicida, previously identified as belonging to the species E. tarda, is a novel species first described from Europe and Asia (Abayneh et al., 2012b) and recently reported from the United States (Griffin et al., 2014). In Finland, E. piscicida has been associated with disease outbreaks in whitefish during different years. The results suggest that whitefish is susceptible to infection with E. piscicida, indicating that this pathogen has a wide geographical distribution and high adaptability for infecting different hosts.

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Since whitefish is a highly valued species in Finland, disease outbreaks due to E. piscicida may be a potential threat for an increased fish production in the future. Acknowledgments We thank the Ministry of Science, Research and Technology of the Islamic Republic of Iran and University of Tehran (21/6/7508002) for the financial support. Maija Liisa Hoffrén and Christine Engblom are acknowledged for technical assistance. References Abayneh, T., Colquhoun, D.J., Sørum, H., 2012a. Multi-locus Sequence Analysis (MLSA) of Edwardsiella tarda isolates from fish. Vet. Microbiol. 158, 367–375. Abayneh, T., Colquhoun, D.J., Sørum, H., 2012b. Edwardsiella piscicida sp. nov., a novel species pathogenic to fish. J. Appl. Microbiol. 114, 644–654. Abayneh, T., Colquhoun, D.J., Austin, D., Sørum, H., 2013. Multi-locus variable number tandem repeat analysis (MLVA) of Edwardsiella piscicida isolates pathogenic to fish. J. Fish Dis. 37. Abbott, S.L., Janda, J.M., 2006. The genus Edwardsiella. 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