Toxicity of Photobacterium Damselae Subsp. Damselae Strains Isolated from New Cultured Marine Fish
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Vol. 92: 31–40, 2010 DISEASES OF AQUATIC ORGANISMS Published October 25 doi: 10.3354/dao02275 Dis Aquat Org Toxicity of Photobacterium damselae subsp. damselae strains isolated from new cultured marine fish Alejandro Labella1, Nuria Sanchez-Montes1, Concepcion Berbel2, Manuel Aparicio2, Dolores Castro1, Manuel Manchado2, Juan J. Borrego1,* 1Department of Microbiology, Faculty of Sciences, University of Malaga, 29071 Malaga, Spain 2IFAPA Centro El Toruño, Junta de Andalucia, Puerto de Santa Maria, 11500 Cadiz, Spain ABSTRACT: The in vivo and in vitro toxicity of bacterial cells and their extracellular products (ECPs) from 16 strains of Photobacterium damselae subsp. damselae isolated from 7 epizootic outbreaks × 5 were evaluated. On the basis of their 50% lethal dose (LD50) values (about 1 10 CFU), these strains may be considered as moderately virulent. However, their ECPs were strongly lethal for redbanded seabream Pagrus auriga causing fish death within 2 h post-inoculation (protein concentration ranged between 2.1 and 6.41 µg g–1 fish). The bacterial ECPs tested exhibited several enzymatic activities, such as amylase, lipase, phospholipase, alkaline phosphatase, esterase-lipase, acid phosphatase, and β-glucosaminidase. These ECPs displayed a strong cytotoxic effect on 4 fish and 2 mammalian cell lines, although this activity disappeared when ECPs were heated at 100°C. The virulence of the strains tested could not be related to the hemolytic activity or to the production of the toxin damselysin. Therefore, another unknown type of toxin could play an important role in the virulence mechanisms of this bacterial pathogen. KEY WORDS: Toxicity · ECP · Photobacterium damselae subsp. damselae · Cultured marine fish Resale or republication not permitted without written consent of the publisher INTRODUCTION tion, growth, and immune system of species such as redbanded seabream Pagrus auriga, white seabream Photobacterium damselae subsp. damselae is a Diplodus sargus, and meagre Argyrosomus regius are halophilic bacterium associated with several diseases ongoing (Cardenas & Manchado 2008, Martin-Antonio affecting both wild and cultured fish species (Fouz et et al. 2009). However, the intensive culture of these al. 1991, 1992, Vera et al. 1991) and mammals, includ- fish species and others has favored the appearance of ing humans (Clarridge & Zighelboim-Daum 1985, Cof- several outbreaks with moderate mortality involving fey et al. 1986, Buck et al. 1991, Yamane et al. 2004). Photobacterium damselae subsp. damselae (Labella et Epizootic outbreaks of this bacterial pathogen have al. 2006, Garcia-Rosado et al. 2007, Kanchanopas- been associated with immunocompromised or stressed Barnette et al. 2009). fish by overcrowding (Thune et al. 1993, Garcia- Several virulence factors are involved in the patho- Rosado et al. 2007). Symptoms of these bacterial genesis of Vibrio and Photobacterium genera (Norqvist pathologies are not specific, including hemorrhagic et al. 1990, David et al. 1992, Toranzo & Barja 1993, septicemia with extensive skin lesions and focal necro- Magariños et al. 1994, Bakopoulos et al. 1997, Perez et sis of the liver, spleen, kidney, and other tissues al. 1998, Farto et al. 2002, 2006, Ishihara et al. 2002, (Hjeltnes & Roberts 1993). Wang et al. 2007). In the case of P. damselae subsp. Several new marine species are being evaluated as damselae only an extracellular cytolytic toxin, named potential candidates for aquaculture production. In damselysin, which is a phospholipase-D active against southern Europe, studies on reproductive cycles, nutri- the sphingomyelin of the sheep erythrocyte mem- *Corresponding author. Email: [email protected] © Inter-Research 2010 · www.int-res.com 32 Dis Aquat Org 92: 31–40, 2010 brane, has been described (Kreger 1984, Kothary & MATERIALS AND METHODS Kreger 1985, Kreger et al. 1987). In addition, a relation- ship between the degree of virulence and the Bacterial strains. In this study, 16 Photobacterium hemolytic activity has been demonstrated in P. damse- damselae subsp. damselae strains associated with epi- lae subsp. damselae strains isolated from fish (Fouz et zootic outbreaks affecting various cultured marine fish al. 1993). However, Cutter & Kreger (1990) found that species (Table 1) were used. All the isolates were not all the P. damselae subsp. damselae strains pre- phenotypically and genotypically characterized as sented the damselysin gene (dly), but only those previously described (Labella et al. 2006, 2009, Kan- strains showing intense hemolytic activity. A further chanopas-Barnette et al. 2009). P. damselae subsp. study demonstrated that the presence of this gene was damselae strain ATCC 33539T, isolated from dam- not correlated to the virulence in mice and fish of selfish, was used for comparative purposes. 17 P. damselae subsp. damselae strains isolated from Strains were routinely cultured on tryptic soy agar or different sources (Osorio et al. 2000). broth (TSA or TSB) (Difco Lab) supplemented with The extracellular products (ECPs) are produced by 1.5% (w/v) NaCl (TSAs or TSBs, respectively), and bacterial pathogens to facilitate the uptake of nutrients incubated at 22°C for 2 to 5 d. Stock cultures were from the surrounding environment, and/or for the suc- stored frozen at –80°C in TSBs with 15% (v/v) glycerol. cessful penetration and survival of pathogens inside ECPs extraction. Bacterial ECPs were obtained by the host (Bakopoulos et al. 2003). However, the role of the cellophane plate technique (Liu 1957) by spread- ECPs in the pathogenesis of Photobacterium damselae ing 3 ml of a 24 h broth culture of each strain over ster- subsp. damselae in fish is poorly known, which is a ilized cellophane sheets placed on TSAs plates. After considerable disadvantage for the development of vac- incubation for 48 h at 22°C, bacterial cells were cines and vaccine strategies, since it has been sug- washed off the cellophane sheets with phosphate- gested that the ECP components are major antigenic buffered saline (PBS) at pH 7.0. The bacterial suspen- compounds of several vaccine formulations (Collado et sions were centrifuged at 13 000 × g for 20 min at 4°C, al. 2000, Bakopoulos et al. 2004). and the respective supernatants were filtered through The aim of this study was to determine the toxicity of 0.45 µm pore-size membrane filters. All the ECP sam- different Photobacterium damselae subsp. damselae ples were stored at –20°C until used. The protein con- strains isolated from cultured diseased fish. For this centration of the ECPs was spectrophotometrically purpose, we performed in vivo and in vitro assays determined by using bicinchoninic acid (Sigma- using bacterial cultures and their ECPs. In addition, Aldrich) and bovine serum albumin (Sigma-Aldrich) as the enzymatic activities of the ECPs and their cyto- standard. To evaluate the total proteolytic activity pre- toxicity in fish and mammalian cell lines were com- sent in the ECP samples, a multiprotein substrate pared. (Azocoll; Sigma-Aldrich) was used following the Table 1. Source of the Photobacterium damselae subsp. damselae strains and protein content and total proteolytic activity of their extracellular products. ATCC: American type culture collection Strain Source Isolation Outbreak Proteins Proteolytic organ date (µg ml–1) activity (U ml–1) 203H Redbanded seabream Pagrus auriga Liver Winter 2003 910.3 0.23 401H Redbanded seabream P. auriga Liver Spring 2004 594.16 0.04 402O Redbanded seabream P. auriga Eye Autumn 2004 412.50 0.59 403D1 Redbanded seabream P. auriga Intestine Autumn 2004 533.05 0.16 412B Redbanded seabream P. auriga Spleen Autumn 2004 473.05 <0.01 9401H Redbanded seabream P. auriga Liver Summer 2004 877.08 <0.01 501H Redbanded seabream P. auriga Liver Summer 2005 613.61 <0.01 501R Redbanded seabream P. auriga Kidney Summer 2005 346.38 <0.01 501B Redbanded seabream P. auriga Spleen Summer 2005 218.05 <0.01 601R Redbanded seabream P. auriga Kidney Summer 2006 414.72 0.05 602R Redbanded seabream P. auriga Kidney Summer 2006 347.50 <0.01 D408U Gilt-head seabream Sparus aurata Ulcers Autumn 2004 835.27 0.93 Lb501R European seabass Dicentrarchus labrax Kidney Summer 2005 564.72 0.12 S503C White seabream Diplodus sargus Brain Summer 2005 554.72 <0.01 C601R1 Meagre Argyrosomus regius Kidney Summer 2006 774.16 <0.01 LCA240907 Barramundi Lates calcarifer Liver Autumn 2007 1282.39 0.38 ATCC 33539T Damselfish Chromis punctipinnis – 420.00 0.02 Labella et al.: Toxicity of Photobacterium damselae subsp. damselae 33 manufacturer’s instructions. An absorbance reading of epithelial cell line. Cells were cultured and maintained 1.0 at 520 nm, after a 30 min assay at 37°C, was defined using Dulbecco’s modified Eagle’s medium (HyClone as one unit of protease activity. Laboratories) supplemented with 4 mM L-glutamine Determination of enzymatic activities. The enzy- (Sigma-Aldrich), bovine calf serum (Gibco) to a final matic activities presented in the ECPs and in bacterial concentration of 10% (v/v) and 1% (v/v) penicillin– cultures were evaluated using the API ZYM system streptomycin solution (Gibco). Cell cultures were incu- (bioMérieux). Other enzymatic activities, such as bated in 5% CO2 at 37°C and 95% humidity. Four caseinase, gelatinase, amylase, phospholipase, and different fish cell lines were tested: SAF-1, a fibroblas- lipase, were determined in plates by a diffusion tic cell line from gilt-head seabream Sparus aurata method. Plate dishes of saline basal nutrient agar (Bejar et al. 1997); BF-2, a fibroblastic cell line from (Difco) (1.5% NaCl, w/v) were supplemented with bluegill fry Lepomis macrochirus; CHSE-214, an epi- 1% (w/v) sodium caseinate (Difco), 1% (w/v) gelatin thelial cell line from Chinook salmon Oncorhynchus (Oxoid), 0.4% (w/v) starch (Difco), 2% (v/v) egg yolk tshawytscha; and SSN-1, an epithelial cell line derived emulsion (Sigma-Aldrich) and 1% (v/v) Tween- from striped-snakehead fish Ophicephalus striatus. 20/Tween-80 (Sigma-Aldrich), respectively. In addi- Fish cells were cultured and maintained using Lei- tion, commercial deoxyribonuclease test medium bovitz medium (L-15; Gibco), supplemented with 2% (Biolife) was used for the DNase test.