Occurrence and expression of virulence-related properties by environmental halophilic Vibrio spp. in in vitro and in vivo systems W. Baffone * , E. Vittoria, R. Campana, B. Citterio, A. Casaroli, L. Pierfelici Istituto di Scienze Tossicologiche, Igienistiche ed Ambientali, University of Urbino, via S. Chiara 27, Urbino 61029, Italy Received 28 January 2004; received in revised form 5 May 2004; accepted 6 May 2004 Abstract The virulence characteristics of 87 strains of halophilic Vibrio spp. from seawater collected near aquaculture areas and seafood products were evaluated by in vitro and in vivo methods. Adhesive properties were revealed in 69% of the analysed strains in Caco-2 cells and in 82.7% when using Hep-2 cells. A weak type of adherence seems to be more common. 19.3% of the strains, prevalently represented by V. alginolyticus, appears to produce cytotoxic effects on HeLa cell line while 8.6% exhibits enterotoxic activity. Five of the six V. parahaemolyticus strains results able to produce a thermostable direct haemolysin (TDH), confirmed as the major virulence factor in the pathogenic mechanism of action of this Vibrio species. Because of the strain-specific pathogenicity of most halophilic Vibrio spp., their detection in seafood, giving particular attention to their pathogenicity, is necessary to improve the control of these products. Ó 2004 Elsevier Ltd. All rights reserved. Keywords: Halophilic Vibrio; Virulence properties; In vivo; In vitro systems 1. Introduction The genus Vibrio now includes a large number of species that are ubiquitous in many environments and can be readily isolated from water, sediments and sea- foods such as oysters, mussels and coastal fish (DePaola, Capers, & Alexander, 1994; Jak si  c, Uhitil, Petrak, Ba zuli  c, & Karolyi, 2002; Kelly & Dan Stroth, 1988a, 1988b; Matte, Matte, Rivera, & Martins, 1994). Clear evidence is available concerning the aetiological role of Vibrio cholerae, V. vulnificus and V. parahaemo- lyticus in food-borne diseases (Feldhusen, 2000). Until recently the role of other Vibrio spp., such as V. algi- nolyticus, V. fluvialis, V. furnissi, V. hollisae, V. mimicus, V. metschnikovii and V. damsela, has not been firmly established although they are implicated as responsible for a smaller, but relevant number of cases (Morris, 1999; Thekdi, Lakhani, Rale, & Panse, 1990), in some of which the infection source is attributed to contaminated seafood (Abbott & Janda, 1994; Dalsgaard et al., 1997; Hillers, Medeiros, Kendall, Chen, & DiMasciola, 2003; Thekdi et al., 1990). The mechanisms by which these halophilic Vibrio provoke disease are still poorly understood. Virulence factors that could potentially play a role in their pathogenesis include the ability to adhere to (Alam, Miyoshi, Yamamoto, Tomochika, & Shinoda, 1996) and invade epithelial cells (Miliotis, Tall, & Gray, 1995) as well as toxin production (Barbieri et al., 1999; Chowdhury, Aziz, Bradford, & Rahim, 1987). The ability to adhere to epithelial cells, a common virulence character in various pathogenic bacteria, is recognised in several Vibrio spp. (Alam et al., 1996) as an auxiliary virulence associated factor, while the ability to invade the host cells is a virulence mechanism sug- gested for some isolates of V. parahaemolyticus (Akeda, Nagayama, Yamamoto, & Honda, 1997) and for V. hollisae (Miliotis et al., 1995). The secretion of several pathogen-related substances, for example cholera toxin-like enterotoxin (Shinoda, 1999; Spira & Fedorka-Cray, 1984), heat-stable entero- toxin (Nishibuchi & Seidler, 1983; Shinoda, 1999), and haemolysins (Honda, Narita, Yoh, & Miwatani, 1987; Shinoda, 1999) has been reported for these pathogens. * Corresponding author. Tel.: +39-0722-350585; fax: +39-0722- 4717. E-mail address: baffone@uniurb.it (W. Baffone). 0956-7135/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodcont.2004.05.006 Food Control 16 (2005) 451–457 www.elsevier.com/locate/foodcont