Short communication Characterization of the virulence of Harveyi clade vibrios isolated from a shrimp hatchery in vitro and in vivo, in a brine shrimp (Artemia franciscana) model system Sofie Vanmaele a , Tom Defoirdt a, ⁎, Ilse Cleenwerck b , Paul De Vos b , Peter Bossier a a Laboratory of Aquaculture and Artemia Reference Center, Ghent University, Rozier 44, 9000 Ghent, Belgium b Laboratorium of Microbiology, Ghent University, K.L. Ledeganckstraat 35, 9000 Ghent, Belgium abstract article info Article history: Received 1 August 2014 Received in revised form 5 September 2014 Accepted 10 September 2014 Available online 16 September 2014 Keywords: Artemia franciscana Shrimp Vibrio spp. Virulence factors Vibrios belonging to the Harveyi clade are important pathogens of a large number of marine animals in the aqua- culture industry. In this study, six isolates (H1 to H6) were obtained from a shrimp hatchery in Rio Grande do Norte (Natal-Area, Brazil), which had been confronted with disease outbreaks in 2009. The aim was to character- ize the virulence of these isolates, both in vitro (virulence factor production) and in vivo (virulence towards gno- tobiotic brine shrimp, Artemia franciscana, larvae) and to compare these characteristics to those of the most virulent pathogen in the brine shrimp model described to date, Vibrio campbellii LMG 21363. Of all 6 isolates, H5 and H6 were found to be the most virulent ones and were therefore selected for further characterization. Iso- late H5 exhibited a similar virulence as the control strain, while H6 exhibited a higher virulence, both in gnoto- biotic and conventionally reared brine shrimp. Both H5 and H6 were motile and produced all of the lytic enzymes tested (hemolysin, caseinase, gelatinase, lipase and phospholipase). Although H6 was the most virulent isolate in vivo, this was not reflected in the highest production of all virulence factors tested. Finally, isolates H5 and H6 were identified to belong to the Harveyi clade of vibrios. © 2014 Elsevier B.V. All rights reserved. 1. Introduction Vibrios are Gram-negative bacteria that are ubiquitous in the marine environment and can be found free-living in the water column, as part of a biofilm, or in association with a host (Thompson et al., 2004). The Harveyi clade of vibrios is a subgroup containing major aquaculture pathogens (Ruwandeepika et al., 2012). Vibriosis causes severe eco- nomical losses in shrimp, finfish and mollusk cultivation worldwide (Austin and Zhang, 2006; FAO, 2012; Defoirdt et al., 2007). The pathogenicity mechanisms of vibrios belonging to the Harveyi clade remain largely unraveled; however some virulence indicators have been described. The infectious cycle of pathogenic bacteria in- cludes entry of the pathogen, establishment and multiplication, thereby causing damage to host tissues and cells, and exit (Donnenberg, 2000). These different steps involve the expression of virulence factors — gene products that allow the pathogens to infect and damage the host (Defoirdt, 2013). The ability to colonize and adhere to host surfaces is an essential step in successful infection of a host, and flagellar motility is thought to enhance the initial interaction of a bacterium with a surface by enabling the cell to overcome negative electrostatic forces (McCarter, 2004). Hence, motility can give an indication of the potential of an isolate to colonize a host. A second important phenotype in coloni- zation of the host is biofilm formation (Costerton et al., 1981). The bio- film formation in vibrios depends on several factors including flagella, pili and exopolysaccharide biosynthesis (Yildiz and Visick, 2009). Exopolysaccharides form a loose slime outside the cell that forms an in- tercellular matrix in biofilms, which enhances the growth and survival of microorganisms by providing access to nutrients and protection from detergents or antimicrobials, predators and drying (Donlan and Costerton, 2002). A third group of virulence factors includes lytic en- zymes, which are produced by many pathogenic bacteria and often play a central role in pathogenesis (Finlay and Falkow, 1997). These enzymes cause damage to host tissues, thereby allowing the pathogen to obtain nutrients and to spread through tissues. Lytic enzymes pro- duced by pathogenic vibrios include hemolysins, proteases (including caseinase and gelatinase), and (phospho) lipases (Defoirdt, 2013). In 2009, 6 Vibrio strains (H1-6) were isolated from a shrimp hatch- ery in Rio Grande do Norte (Natal-Area, Brazil), where high mortalities were faced at that moment. In this study, the aim was to characterize the virulence of these isolates, by determining the production of some important virulence factors in vitro, and by determining their virulence towards gnotobiotic and conventionally reared brine shrimp (Artemia Aquaculture 435 (2015) 28–32 ⁎ Corresponding author at: Laboratory of Aquaculture & Artemia Reference Center, Rozier 44, B-9000 Gent, Belgium. Tel.: +32 9 264 37 54; fax: +32 9 264 41 93. E-mail address: Tom.Defoirdt@UGent.be (T. Defoirdt). http://dx.doi.org/10.1016/j.aquaculture.2014.09.015 0044-8486/© 2014 Elsevier B.V. All rights reserved. Contents lists available at ScienceDirect Aquaculture journal homepage: www.elsevier.com/locate/aqua-online